Patient selection

In this study, we chose antiretroviral treatment-naive HIV1 seropositive patients (CD4+ T cell count 3) between the ages of 20 and 60 with or without HCMV co-infection who visited the ART center of designated metropolitan hospitals within 2 years. The primary objective was to identify HIV1-seropositive patients co-infected with HCMV (PCR positive) with either HCMV-induced end-organ retinitis or gastroenteric disease at the time of admission. This constitutes our main target population with a total of 26 individuals collected over a period of one year (15 with HCMV-induced retinitis and 11 with HCMV-induced gastroenteric disease). Two other groups, each containing 25 individuals, were chosen as controls. The groups were divided as follows; Group 1 (HIV1-HCMV co-infected patients who developed end-organ retinitis or gastrointestinal disease), Group 2 (HIV1-HCMV co-infected patients without any symptomatic end-organ disease), Group 3 (HIV1 infected patients without active HCMV infection).

Integration criteria

All HIV1 positive patients were chosen by doctors after carefully monitoring their health status, medical reports and clinical profile. To define “definite HCMV end-organ disease” as retinitis and gastroenteric disease, the presence of appropriate clinical symptoms and/or signs has been confirmed along with documentation of HCMV in the tissues of the affected organ by histopathology, virus isolation, immunohistochemistry or DNA PCR. HCMV-positive patients were identified by amplifying HCMV DNA in blood by PCR. Molecular diagnostics of other opportunistic viruses such as HSV, HBV, HCV, HAV and EBV as well as bacterial, fungal and protozoan cultures etc. were performed and only patients with all-negative results were selected in this study to avoid clinical contradiction.

Exclusion criteria

Patients who died during the follow-up period or for whom a full set of follow-up data was not available were also subsequently excluded. Moribund patients were excluded from the study.

Sample collection and patient data collection

5–10 ml of EDTA (ethylenediaminetetraacetic acid) anticoagulated peripheral blood was collected from patients in Vacutainer tubes, processed immediately, and serum was separated by centrifugation (1000 ×g for 10 min).

All patients were screened and individual patient data was collected by a dedicated physician directly linked to this study. Case notes, charts, survey results, and treatment records for these patients were retrospectively reviewed and statistically analyzed by a verified statistician.

DNA isolation from blood

The QIamp DNA blood Mini kit (Qiagen Inc., Hilden, Germany) was used according to the manufacturer’s protocol to isolate whole DNA from collected blood serum. DNA concentration was measured by measuring OD values ​​using a spectrophotometer.

Qualitative PCR for the detection of HCMV

Primers were designed to amplify the UL 83 and gB regions of the HCMV genome using Primer 3 online software and using standard HCMV strain sequences as reference. Primers were obtained from Eurofins Genomics India Pvt. ltd. The forward and reverse primers for UL 83 were 5′-GGG ACA CAA CAC CGT AAA GC-3′ and 5′-GTC AGC GTT CGT GTT TCC CA-3′. The forward and reverse primers for UL 55 (gB) were 5′-GGTCTTCAAGGAACTCAGCAAGA-3′ and 5′-CGGCAATCGGTTTGTTGTAAA-3′. The detailed procedure was performed according to the protocol described in one of our laboratory’s previously published studies.³⁰.

Cytokine ELISA

Serum TGFβ, IL10, TNFα, IL-6, CRP, IFNγ, IL1β, IL7, MCP1, MIP1α and CXCL10 (IP10) were measured using enzyme immunoassay technique kits from G-Biosciences, Geno Technology Inc., USA . All of these tests were designed to detect only human proteins, even at very low concentrations. The manufacturer’s protocol was strictly followed when performing each assay.

Quantification of HCMV viral load

A standard curve of quantitation was obtained using six ten-fold serial dilutions of an HCMV DNA standard with known viral load (copies/ml) purchased from the ATCC. A conserved partial region of the HCMV UL 75 gene (gH) was amplified in each case and the Ct value was measured in a real-time PCR instrument (ABI 7500-Applied Biosystems). The detailed procedure was performed according to the protocol described in one of our laboratory’s previously published studies.³⁰.

Real-time RNA isolation and polymerase chain reaction

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from whole blood collected using standard isolation instructions. The isolated RNA was converted into cDNA following the standard procedure using primescript 1st Strand cDNA Synthesis Kit (DSS TAKARA Bio India Ltd.). 2 μl of cDNA was used for amplification in a total volume of 20 μl of real-time PCR reaction mix with SYBR green dye (TB Green premix ex taq, DSS TAKARA Bio India Ltd.). The amount of input cDNA was standardized by detecting GAPDH transcripts serving as an internal control in the real-time PCR assay. Relative quantification was performed by determining real-time expression levels of specific mRNAs using the 2^−ΔΔCt method. Primer-3 software was used to manually design the real-time primers in the laboratory and the primers were obtained from IDT technologies INC, India. The reaction conditions and necessary parameters have been standardized in the laboratory and have already been published previously³⁰. All primers that were used in this study for real-time expression have been listed in Supplementary Table 1.

statistical analyzes

Unpaired t-tests and one-way analysis of variance (normal distribution) were used to compare group differences. All results were expressed as mean ± standard deviation, unless otherwise specified. When the distributions were not normal, nonparametric analyzes were performed. Comparison between individual groups was performed using Bonferroni’s method with ANOVA. Statistical significance between two groups was assessed by binary logistic regression. The significance level was set at 5% in all cases. All P values ​​were two-sided. SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA) was used to perform all statistical analyses. Graph Pad Prism software (ver.6) was used to prepare all graphs.

Gene sequencing

Sequencing primers were designed to partially amplify a conserved region of the HCMV UL 115 (gL) gene using Primer 3 software from 22 clinical samples belonging to group 1 (11 patients with retinitis and 11 patients with gastroenteric disease). the primers are: 5′-GTGAGGTGTTTCAGGGGTGACAAGTATG-3′ and 5′-ATTCCTTCACTGCGTTGTACAGGC-3′ respectively. Primers were obtained from Eurofins Genomics India Pvt. ltd. A thermal cycler was used to amplify the specified region. These raw PCR products were outsourced to Agrigenome Labs Pvt. Ltd for Sanger sequencing. After obtaining the sequences, they were analyzed and submitted to the NCBI DNA Databank via sequin (GenBank accession numbers MT263152 to MT263173).

Phylogenetic analysis of HCMV gL gene sequences

22 nucleotide sequences corresponding to a conserved region of the HCMV gL gene were obtained from the HCMV group 1 clinical strains (11 from patients with retinitis and 11 from patients with gastrointestinal disease. 12 complete nucleotide sequences of gL belonging to different geographical locations were extracted from the NCBI RefSeq database as reference sequences (available at https://www.ncbi.nlm.nih.gov/). Nucleotide sequences were aligned using MUSCLE³¹ and misaligned regions with more than 20% deviations were trimmed using trimAl³². After that, a phylogenetic analysis was performed based on the maximum likelihood (ML) method. Bootstrap analyzes were performed with 1000 replicates to determine the robustness of individual nodes in the phylogenetic tree. Evolutionary analyzes were conducted in MEGA-X³³.

Codon Adaptation Index (CAI) Analysis

The Codon Adaptation Index (CAI) is a quantitative measure that predicts the highest relative adaptation of viruses to their potential host. CAI is calculated using the CAI programmer available in the EMBOSS package (http://www.bioinformatics.nl/cgi-bin/emboss/cai)³⁴. The reference dataset for Homo sapiens were retrieved from the Codon Usage Database (http://www.kazusa.or.jp/codon/).

Analysis of the effective number of codons (ENc)

The degree to which codon usage deviates from random selection is defined by the ENc value. The value also describes the degree of preference for non-equilibrium usage of synonymous codons within the codon family. The ENc value was calculated using the chip program available in the EMBOSS package (http://www.bioinformatics.nl/cgi-bin/emboss/chips)³⁵.

Analysis of mRNA structure of HCMV gL genes

Here, we analyzed the secondary structure of the potentially conserved local mRNA of the partially sequenced region of the gL gene and compared the structural patterns between the two groups of clinical strains. LocARNA was used to generate local structural alignment and consensus structure of gL gene sequences from clinical and reference strains^36.37.

Ethical considerations

This study and methodologies have been approved by the Scientific Advisory Committees (SAC) and certified by the Institutional Ethics Committee (IEC) of ICMR NICED, Kolkata as well as all associated hospitals in accordance with the Declaration of Helsinki of 1964.

Patient Consent

Written informed consents were collected after explaining all positive and negative aspects associated with the study to each participating patient in languages ​​including at least one that they clearly understand. Confidentiality of the medical and clinical information provided was properly maintained in accordance with standard guidelines.