Seroprevalence and Biomarkers Detection of Human Herpes Virus (HHV-8) in Patients with Kaposi Sarcoma

Abstract


Introduction
Kaposi sarcoma (KS) is caused by the human Herpesvirus (HHV)-8, in addition to the unique primary effusion lymphoma [1]. Sexual contact can lead to HHV-8 transmission [2] and via nonsexual ways [3]. Although some scientists have discovered viral DNA in sperm as well as normal and malignant prostate tissue [4], other scientists found no viral DNA in the above-mentioned tissues [5]. As HHV-8 is able to cause cancer, is able to be sexually-transmitted, and was found in prostate tissues by certain researchers, then it can be a viable option for infectious agent cofactor [6].
The epithelial cell lines of the 293 human kidney tissue were the most vulnerable [7]. CD19+ B-cells are HHV-8's essential biological repositories [8]. Endothelium has been found to be naturally infected in different cell types [9], monocyte [10], prostate glandular epithelium [11], sensory ganglion cells of the dorsal root [12], as well as spindle cells from Kaposi sarcoma tumors [9].
HHV-8, like other rhadino viruses, may be harmful only in the presence of additional cofactors, e.g. the concurrent HIV infection or the immunocompromised hosts. HHV-8is largely harmless in the natural healthy host [13]. Currently, however, there is really no recognized host else than humans. HHV8, that belonged to genus Rhadinoviridae within subfamily Gammaherpesvirinae , uses B-lymphocytes as cell reservoirs. Thus, many studies have used lymphoid cell lines (such as BC3 and BCBL1) from patients with pleural lymphoma as a viral source for research into the effects of phorbol ester, 12-o-tetradecanoylphorbol-13-acetate, (TPA), that triggers the lysis phase and the later creation of particles of the infectious virus. HHV8 shows no clear cytolyticimpactat the lysis phase and thus, molecular methods are required for its detection (such asamplication of PCR for specific genes like latency factor LANA, or immunostaining of surface antigens K8.1) [14], Human umbilical vein endothelial cells (HUVEC) and the BAC36 strain were used to provide a highly effective cellular model of infection and viral lytic replication. Such a model allows for defining the lytic-latent phase, revealing that viral production begins at 1-2 days and continues to 10-15 days after infections, followed by its entering latent states as episomes bound with cellular DNA-associated histones where it can remain for the complete lifespan of hosts [15]. HHV8 causes considerable changes in cellular biochemistry and physiology during latent infection, involving changes in HHV8 permeability of cells, resistance to toxic medicines, increased resistance to stressful conditions, enhancement of glycolysis as well as higher expressions to the insulin receptor (IR) [16]. All of the acquired properties and conditions may result in oncogenesis and cell transformations like KS and neoplastic inductions of lymphoma cells. Unfortunately, Nucleoside analogue acyclovir and other standard anti-Herpes medications are ineffective against HHV8 during the latent phase and do not prevent HHV8 development during the latent phase  [17].
If HHV8 is a co-factor in of prostate cancer progression, then HHV8 sero-prevalence in males with disease may be higher than in age-matched controls. In the current study, we determine the seroprevalence and biomarkers detection of Human Herpes Virus (HHV-8) in Iraqi patients with sarcoma. Serum markers' involvement and prognostic importance in uterine carcinosarcomas (CSs).
The purpose of this study was to see if serum CA 125, CA 15-3, in addition to CA 19-9 might be used as prognostic indicators and as markers for illness progression in individuals with CS.

Sample collection
Blood samples were taken for this study from 120 subjects, where 60 of them were positive to HHV-8 with sarcoma and 60 of the subjects are healthy volunteers. Blood samples were collected from patients who attended Baghdad Teaching Hospital from the period of 15th February 2021 to 15th January 2022.

DNA extraction
DNA extraction was performed as described by Guech-Ongey, (2008) with slight modification [18]. In brief, about 0.2ml of blood sample was digested with proteinase K (150μg/ml for 45min at 60°C) in lysis buffer (10mM Tris-HCl pH 7.6, 5mM EDTA, 150mM NaCl, and 1% SDS). The contents are then centrifuged at maximum speed for 10min and the supernatant was collected into a fresh tube. DNA was now purified with phenol and phenol-chloroform-isoamyl alcohol (25: 24: 1) and extracted with ethanol.

HHV8 Real-Time PCR
Each DNA sample's HHV8 viral load was measured using SYBR Green real-time PCR as described by Sudhakar and Raman, (2020) but with slight modifications [20]. Primers studied before [21] were used in the study and ordered from Sigma Aldrich. ORF26LR1FW (5′-GCAGTATCTATCCAAGTG-3′) and ORF26LR2RV (5′-ACAGATCGTCAAGCA-3′) were used and found to produce 434bp product (Table 1). HHV8 viral quantification was done in the Ependorf Real-time PCR Detection System with 200ng of template DNA. About 12.5μL of SYBR Green supermix (Himedia), and 5 pmol each of forward and reverse primers were used and the total volume was made to 25μl. DNA was amplified using the following steps: initial 3min denaturation at 95°C, 50cycles of 55°C annealing for 30sec, 72°C extension for 30sec and a final 5min elongation at 72°C. All the experiments were done in triplicates and the human β-globin gene (FW5′-GAAGAGCCAAGGACAGGTAC-3′, and RV 5′-CAACTTCATCCACGTTCACC-3′) was used as a housekeeping gene to normalize the target DNA.

Sequencing and phylogenetic analysis
Eurofins Genomics (Bangalore, India) used fluorescent dye terminator technology and ABI 3730 DNA sequencers to perform bidirectional direct sequencing on the obtained PCR products (Applied BioSystems, Foster City, CA). Sequences obtained in FASTA format were studied by clustal W to depict the phylogenetic relation to the reference sequences. Reference sequences used in the study were DQ984689.1 and DQ984768.1 (HHV8 ORF26 subtype).

Statistical analysis
The SPSS-20 software program (Faculty version) was used for statistical analysis of data, involving the t-test. (P<0.05) value was regarded as statistically significant. Table 2 showed the infection distribution according to gender. The number and percentage of males are 33 (55.0%), and females were 27(45.0%). There was no significant difference obtained in the number based on gender as observed (chi-square 0.03; P=0.8).  We found Ca19.9 biomarker was significantly observed within the subjects when compared to control (P<0.05). Table 4 showed the distribution of Ca19.9 biomarker in patients with HHV-8 infections wherein we could see that 10(50.0%) of the 20 infected patients were positive with Ca19.9 (P-value =0.03). We found no significance in the expression of Ca19.9 biomarker within the subjects when compared to control (P=0.3). Table 5 showed the distribution Ca125 biomarker in patients with HHV-8 infections explained that 5(25%) of 20 infected patients were positive with Ca125, Pvalue =0.3. We found Ca15.3 biomarker was significantly observed within the subjects when compared to control (P=0.001). All of the 60 samples showed positive amplification for the HHV8 ORF26 gene. A PCR product of 434bp was seen among all the samples (n=60 positive) which was sequenced. Only 20 samples were sequenced and the results were analyzed using MEGA 7. The phylogenetic tree (Fig. 1) showed a close relation between the samples and all the samples were confirmed to be HHV8 irrespective of their regions.

Discussion
In humans, herpesvirus (HHV8) causes Kaposi sarcoma (KS) and therefore a rarer primary effusion lymphoma. The distribution of HHV8 infections according to the gender was illustrated the male was 33 (55.0%) and the female 27(45.0%).These findings agreed with (Tembo et al., 2017) who reported that the mostly males (64.4%, 56/84) than females (33.3%, 28/84) [25]. These findings agreed with Mamimandjiami et al., 2021 who found that males more than females were infected due to human herpes virus (HHV8) [26]. Also Chavoshpour-Mamaghani et al., (2021) reported that male was higher significant affected than female in humans, herpes virus (HHV8) [27].  [28]. Shakir et al., (2018) proved that no significant differences among ages with humans, herpes virus (HHV8), P<0.05. In humans, herpes virus (HHV8) is one of the most dangerous types that may lead to death. Recently, many cases of death have been recorded when it hits organs in the human body such as the heart and liver, and this leads to death because of its transmission to those organs without symptoms. This virus weakens the immune system and causes great damage, and may help in the programmed killing of the cells of those organs, as it is a silent killer [29]. The distribution Ca19.9 biomarkers in patients with HHV-8 infections explained that 10(50.0%) of infected patients were positive with Ca19.9. CA 19.9 rise is not associated with neoplastic illness. Ca19.9 marker levels are increased in the sera of patients with pancreatic cancer, as it is an indication of tumor occurrence. These results are used with histological and PET SCAN examinations, as they may be an important guide for the physician in the initial diagnosis. And the high levels of Ca19.9 is a marker not only for determining the incidence of pancreatic tumors, but perhaps there are neighboring organs that have been invaded by these tumors, such as the liver, so this indicator is of great importance in determining these cancers [30]. Lyu et al., (2021) has been proven a high percentage in levels of CA19.9 for patients with pancreatic tumors and that this indicator is of great importance in determining glandular tumors, especially pancreatic tumors. While in the benign tumors of the pancreas, there were average levels in the proportions of measurements of CA19.9 [31]. Poruk et al., (2013) explained there is a high accuracy and sensitivity in using the CI 19.9 marker in determining the incidence of pancreatic cancer compared to benign tumors [32]. The distribution Ca125 biomarker in patients with HHV8 infections explained that 5(25.0%) of infected patients were positive with Ca125. Funston et al., (2020) explained that the CA125 values are provided that correspond to the nearest integer probability of 3%. The probabilities, which correspond to a CA125 level of 35 U/ml, are also indicated [33]. Also Edula et al., (2018) agreed with the report of Funston et al., (2020) [34]. The distribution Ca15.3 biomarkers in patients with HHV8 infections explained that 16(80.0%) of infected patients were positive with Ca15.3. These results agreed with (Shakir et al., 2018) who proved in terms of the tumor marker CA15-3, one patient (7.1%) tested positive for HHV8 antibodies. In terms of the PR, ER (expression of estrogen receptor (ER), progesterone receptor (PR)), and HER2/neu, 9 of the 45 patients were tested, and no instances positively tested for HHV8 IgG antibodies. Statistically, the discrepancies between the HHV8 IgG antibodies and the various tumor markers were significant [29]. But Chu et al., (2016) disagreed with present study and reported no significant effect of CA15-3 in HHV8 infection. High levels of CA15-3 in the sera of patients suspected of being blind, because breast cancer is the most common because this marker is one of the indicators of the occurrence of tumors in the human body. In addition, the measurement of this indicator can be useful for those who suffer from common injuries, especially women; chest tumors may be associated with other sites of cancer cervical tumors [35]. The existence of HHV8 DNA was verified by running nested-PCR product on a 1.5 percent agarose gel with a product size of 233bp. Tembo et al., (2017), his research stated that genes are determined with the extracted DNA was utilized to detect the HHV8 sequences. The ORF26 gene KS-1 (5'-AGCCGAAAGATTCCACCAT-3') and KS-2 (5'-TCCGTGTTGTCTACGTCCAG-5'), KS-4 (5'-CGAATCCAACGGATTTGACCTC-3') and KS-5 (5'-CCCATAAATGACACATTGGTGGTA-3') primers were used to amplify the HHV-8 genomes [25]. Ramezani et al., (2016) proved that similar product with primers were used with HIV infection [36].

Conclusions
We concluded that Ca19.9 biomarkers, Ca 125 biomarker and Ca 15.3 biomarker gave positive results in patients due to Human Herpes Virus 8 (HHV-8) infections. In conclusion, HHV8 DNA sequences are detected in a substantial proportion of HIV-positive patients. The potential of the amplified gene segment as a molecular diagnostic tool has to be investigated in greater depth.