Rotavirus and adenovirus in children evaluated for viral gastroenteritis at a single healthcare center in the Eastern Province of Saudi Arabia: A perspective of two decades.

BACKGROUND: The aim of this study was to determine the distribution of rotavirus and adenovirus in pediatric patients evaluated for viral gastroenteritis in a hospital in the Eastern Province of Saudi Arabia for 22 years. MATERIALS AND METHODS: This was a retrospective study based in a secondary healthcare center in Saudi Arabia. Laboratory and demographic data were collected from hospital records for all pediatric patients (up to 14 years old) evaluated for viral gastroenteritis by rotavirus/adenovirus antigen detection kit from January 2000 to December 2022. Data were analyzed utilizing SPSS version 28.0. Categorical data were presented as frequency and percentages, whereas mean and standard deviations were computed for continuous variables. Chi-square test and t-test were used to determine statistical significance. RESULTS: The overall yields of antigen detection were 13.6% for rotavirus and 2.6% for adenovirus. Coinfection with both viruses was documented in 0.5% of the study population. Rotavirus was persistently detected in the past two decades with varying frequency, but the detection of adenovirus showed intervals of at least three consecutive years of zero confirmed cases. Before 2013, when the rotavirus vaccine was introduced in Saudi Arabia, rotavirus was much more prevalent than adenovirus (30% compared to 3.8% in 2010), but they became equally prevalent a decade after the introduction of the vaccine. Rotavirus gastroenteritis showed three different peaks in the year, in March, July, and December. Each peak was followed by a gradual decrease in prevalence before the next peak. Adenovirus, in contrast, was detected consistently around the year at rates between 2% and 5%. CONCLUSION: Rotavirus and adenovirus gastroenteritis have changed in prevalence in the past two decades. We found distinct seasonal patterns associated with rotavirus and adenovirus gastroenteritis. The utilization of virological testing for pediatric gastroenteritis with syndromic testing panels is to be encouraged to improve the knowledge of the true prevalence of enteric viruses.

Low levels of soluble DPP4 among Saudis may have constituted a risk factor for MERS endemicity.

Most of the cases of Middle East respiratory syndrome coronavirus (MERS-CoV) were reported in Saudi Arabia. Dipeptidyl peptidase-4 (DPP4) was identified as the receptor for the virus. The level of soluble DPP4 (sDPP4) was found to be reduced in MERS-CoV infected patients while high levels of sDPP4 were suggested to be protective against MERS-CoV in animal models. We investigated whether the Saudi population has lower levels of sDPP4 which makes them more susceptible to MERS-CoV infection and, therefore, could explain the larger number of cases from the country. Blood samples were collected from 219 Saudi blood donors and 200 blood donors from other ethnic groups. The plasma level of sDPP4 was measured by ELISA and the following SNPs in the DPP4 gene; rs35128070, rs1861978, rs79700168, and rs17574, were genotyped by TaqMan SNP genotyping assay. The average level of plasma sDDP4 was significantly lower in Saudis than other Arabs and non-Arabs (P value 0.0003 and 0.012, respectively). The genotypes AG of rs35128070 and GT of rs1861978 were significantly associated with lower sDPP4 among Saudis (P value 0.002 for each). While both genotypes AA and AG of rs79700168 and rs17574 were associated with significantly lower average sDPP4 level in Saudis compared to other ethnic groups (P value 0.031 and 0.032, and 0.027 and 0.014, respectively). Herein, we report that the Saudi population has lower levels of plasma sDPP4 than other ethnic groups, which is associated with genetic variants in the DPP4 gene. This may have contributed to increase the susceptibility of the Saudi population to MERS-CoV infection and could be a factor in the long-lasting persistence of the virus in the country.

Assembly of infectious Kaposi's sarcoma-associated herpesvirus progeny requires formation of a pORF19 pentamer.

Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its ß-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases.

Polymorphic Variants in 5'-UTR Regions of the RAD51 Gene are Associated With RAD51 Expression and Triple-Negative Breast Cancer (TNBC): A Case-Control Study.

Breast cancer is a heterogeneous disease at morphologic and molecular levels, which is considered the most commonly occurring cancer in women. RAD51, a DNA-repairing protein, involves homologous recombination and has a vital role in genome stability. Polymorphism of the RAD51 gene, and its overexpression, has been proposed to be associated with the development of breast cancer. Overexpression of RAD51 in many types of human cancer including metastatic breast cancer may signify its potential use as a biomarker. Considering the numerous reports on the role of the 5'-UTR-RAD51 polymorphism in breast cancer, this study aimed to investigate the utility of RAD51 gene expression and its variants G135C and G172T as a possible foretelling factor of breast cancer development. DNA sequencing and immunohistochemistry of RAD51 were conducted on 103 samples from patients diagnosed with sporadic breast cancer and 80 samples from a control group. The results demonstrated that the RAD51 variants, G135C and G172T, were significantly presented in the breast cancer tissue compared with the control group. RAD51 expression was mainly shown in the cytoplasm of malignant cells (56% of cases) and significantly correlated with p53 and G135C, C135C variants. Moreover, the occurrence of the G172T variant was significantly associated with the expression of estrogen receptor. Interestingly, 21/26 (81%) of the triple-negative breast cancer showed G135C and C135C genotypes that were significantly associated with the expression of RAD51 (73%). In conclusion, the G135C and C135C variants together with the cytoplasmic expression of RAD51 may have clinical potential as a prognostic predictor for breast cancer development and aggressiveness.

Investigations of a Possible Role of SNPs in KAI1 Gene on Its Down-Regulation in Breast Cancer.

OBJECTIVE: KAI1 (CD82) is a metastasis suppressor gene known to be down-regulated in carcinomas of breast, prostate and many other organs. The mechanism of KAI1 down-regulation is complex and not well understood. Here, we investigate the role of 8 SNPs (not previously studied) in KAI1 gene that could influence its expression in tumor tissue samples of breast cancer patients from the Eastern province of Saudi Arabia. METHODS: Single nucleotide polymorphisms (SNPs) in KAI1 gene were selected from the NCBI website (dbSNP) and were then filtered for those SNPs causing stop codon mutations (rs139889503 and rs150533529) or nonsynonymous mutation in the 5'-UTR (rs11541048, rs77359459, rs115500759, rs182579675, rs200238062, and rs372733853). SNPs genotyping was performed using TaqMan SNP Genotyping Assay and the results were correlated with KAI1 protein expression profile by immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) samples of breast cancer and control none-neoplastic tissues. RESULTS: KAI1 expression by IHC was observed in all none-neoplastic breast tissue samples and only in 35% out of the 59 breast cancer tissue samples. None of the samples was homozygous for the stop codon allele A in the SNP rs139889503 or allele T in the SNP rs150533529. The SNPs in the 5-UTR, rs11541048, rs115500759, and rs182579675, were only present in the homozygous state for the G and C alleles respectively in both cancer and control samples. The other SNPs in the 5'-UTR (rs77359459, rs200238062, and rs372733853) had no significant difference in the allele distribution between KAI1 expressing or none-expressing tissue samples. CONCLUSION: Our findings showed no significant effect of the studied SNPs on down-regulation of KAI1 expression.
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Pathway-Affecting Single Nucleotide Polymorphisms (SNPs) in RPS6KA1 and MBIP Genes are Associated with Breast Cancer Risk.

BACKGROUND: Genetic mutations and polymorphisms play an important role in the transformation of primary cells to malignant cells as it may lead to disturbance of vital pathways regulating cell cycle, DNA damage repair, and apoptosis. In this study, we genotyped single nucleotide polymorphisms (SNPs) which were predicted to affect certain pathways and to increase the risk of breast cancer. METHODS: The study included 81 Saudi breast cancer patients and 100 matching healthy controls from the Eastern Province in Saudi Arabia. The following SNPs (rs3168891, rs2899849, rs2230394, rs2229714) were then genotyped by TaqMan genotyping assay and the allele and genotype distribution was compared. RESULTS: The minor allele frequency of the following SNPs (rs3168891, rs2899849, rs2230394, rs2229714) was T=0.17, A=0.28, A=0.22, and G=0.16 respectively. The G allele of the SNP rs3168891 was significantly associated with increased breast cancer risk (P = 0.00001) while the T allele of the same locus was associated with reduced risk of breast cancer in both heterozygous and homozygous states. The T allele of SNP rs2229714 which is located in the RPS6KA1 gene was also significantly associated with the increased risk of breast cancer. However, the rs2899849 SNP located in the Integrin beta-1 (ITGB1) gene was not associated with the increased risk of breast cancer in our study population. Haplotype analysis revealed the presence of three risk haplotypes that increases the risk of breast cancer (TGGT, TGTA, GATA). CONCLUSION: We showed that three, previously untested, SNPs are associated with increased risk of breast cancer in our population.  This may be added to the list of factors involved in breast cancer risk assessment studies. The benefit and the utility of the in-silico prediction of disease risk factors and their genetic association had been demonstrated in this study, yet the predicted risk alleles have to be tested in clinical studies.
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Alternative splicing is an important mechanism behind KAI1 loss of function in breast cancer patients from Saudi Arabia.

PURPOSE: KAI1 (also called CD82) is a metastasis suppressor gene known to be downregulated in breast cancer and other solid tumors. The downregulation of KAI1 or loss of its function is usually associated with bad prognosis. The mechanism behind KAI1 loss of function is complex. In this study, we investigated "alternative splicing" as a possible mechanism that underlies KAI1 loss of function in breast cancer patients from a tertiary hospital in Saudi Arabia. METHODS: Expression of KAI1 was studied in FFPE breast cancer and control tissue sections by IHC using two different antibodies targeting different domains of the protein. The TS82B antibody targets the extracellular loop, which constitutes most of the protein, while the second EPR4112 antibody targets the C-terminal intracellular domain of the protein. RESULTS: Out of 90 breast cancer samples, 67% showed loss of KAI1 expression. The remaining 33% showed KAI1 expression with (TS82B) antibody; however, the protein was detected in only 11% of cancers when using the antibody (EPR4112) indicating a truncation of the protein at the C-terminus (truncated-KAI1) in 22% of the studied cancer samples. A significant correlation was found between truncated-KAI1 expression and advanced cancer stage (association with lymph node metastasis, P value 0.008). CONCLUSION: Alternative splicing is an important mechanism underlying KAI1 loss of function in breast cancer, and it is associated with bad prognosis (advanced cancer stage).

VEGF Upregulation in Viral Infections and Its Possible Therapeutic Implications.

Several viruses are recognized as the direct or indirect causative agents of human tumors and other severe human diseases. Vascular endothelial growth factor (VEGF) is identified as a principal proangiogenic factor that enhances the production of new blood vessels from existing vascular network. Therefore, oncogenic viruses such as Kaposi's sarcoma herpesvirus (KSHV) and Epstein-Barr virus (EBV) and non-oncogenic viruses such as herpes simplex virus (HSV-1) and dengue virus, which lack their own angiogenic factors, rely on the recruitment of cellular genes for angiogenesis in tumor progression or disease pathogenesis. This review summarizes how human viruses exploit the cellular signaling machinery to upregulate the expression of VEGF and benefit from its physiological functions for their own pathogenesis. Understanding the interplay between viruses and VEGF upregulation will pave the way to design targeted and effective therapeutic approaches for viral oncogenesis and severe diseases.

A Germline Mutation in the BRCA1 3'UTR Variant Predicts Susceptibility to Breast Cancer in a Saudi Arabian Population

Purpose: The impact of the BRCA1-3'UTR-variant on BRCA1 gene expression and altered responses to external stimuli was previously tested in vitro using a luciferase reporter assay. Its ability to predict breast cancer risk in women was also assessed but the conclusions were inconsistent. The present study concerns the relationship between the BRCA1-3'UTR germline variant rs8176318G>T and susceptibility to Breast cancer in an ethnic population of Saudi Arabia. Methodology: The study included 100 breast cancer patients and 100 sex matched healthy controls from the northwestern region (Tabuk) and Dammam of Saudi Arabia were investigated for the BRCA1-3'UTR germline variant rs8176318G>T using an allele specific PCR technique. Genotype distributions were then compared. Results: The frequencies of the three genotypes GG, TT and GT in our Saudi Arabian patients were 26%, 8% and 66% and in healthy controls were 45%, 5% and 50%, respectively (p=0.03). Risk of developing breast cancer was found to be significantly associated with the GT variant (OR 2.28, 1.24-4.191; RR 1.47, 1.11-1.93; P=0.007), GT+TT (OR, 2.32, 1.28-4.22; RR 1.48, 1.13-1.94; P=0.005) and the T allele (OR 1.62 , 1.072- 2.45; RR 1.28, 1.02-1.60: P=0.020). There were 2.76 and 2.28 fold increase risks of developing breast cancer associated with the TT and GT genotypes in our cases. A significant correlation was also found between the BRCA1 3'UTR variants with the stage of the disease and distant metastasis but not with age, grade, and ER, PR and her2/neu status. Conclusion : The rs8176318G/T in the 3'untranslated region (UTR) of the BRCA1 gene was found to be associatedwith increased susceptibility to breast cancer in our study population, increased risk being noted with the GT and TT genotypes. Further association studies are needed to confirm this finding in other regions of Saudi Arabia.

Prevalence of Mouse Mammary Tumor Virus (MMTV)-like sequences in human breast cancer tissues and adjacent normal breast tissues in Saudi Arabia.

BACKGROUND: Breast cancer is considered the most common cancer in women worldwide and is the leading cause of cancer mortality. Sequences similar to Mouse Mammary Tumor Virus (MMTV) were detected in human breast cancer in several studies from different geographical areas. However, the role played by this virus in breast cancer tumorigenesis is not completely understood. These MMTV-like sequences were found to be associated with breast cancer of more malignant types. The aim of this study is to determine the prevalence of MMTV-like envelope gene (env) positivity in breast cancer and non-cancerous breast tissue from Saudi Arabia. METHODS: Detection of MMTV-like env proviral sequences was done using newly designed primers for conventional polymerase chain reaction (PCR). One hundred nighty four samples were collected from 103 females with breast cancer in addition to 51 control breast tissue obtained from individuals without cancer. We additionally investigated the association of proviral positivity with age of the patients, grade of breast cancer and presence of lymph node metastasis. The results were confirmed by sequencing. RESULTS: The prevalence of MMTV-like env proviral positivity was 8.7% (9/103). MMTV env proviral sequences were detected in 5.9% (6/101) of breast cancer tissues and 9.7% (9/93) of non-cancerous adjacent tissues obtained from the same patients. None of the 51 control sample showed positive result for the MMTV env gene. No significant association was found between detection of the virus and the age of the patient, grade of the cancer or presence of metastasis. CONCLUSION: We document the presence of low frequency of MMTV env provirus sequence among breast cancer patients from Saudi Arabia. Further studies are needed to explore the role of the MMTV in breast cancer.

A role for the internal repeat of the Kaposi's sarcoma-associated herpesvirus latent nuclear antigen in the persistence of an episomal viral genome.

The latent nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the replication and partitioning of latent viral genomes. It contains an extended internal repeat (IR) region whose function is only incompletely understood. We constructed KSHV genomes lacking either LANA (KSHV-ΔLANA) or the IR region of LANA (KSHV-LANAΔ329-931). Although still capable of replicating a plasmid containing a latent origin of replication, LANAΔ329-931 does not support the establishment of stable cell lines containing a KSHV genome. These findings suggest a role for the LANA IR in KSHV episomal maintenance without its being required for replication.

Deletion of Kaposi's sarcoma-associated herpesvirus FLICE inhibitory protein, vFLIP, from the viral genome compromises the activation of STAT1-responsive cellular genes and spindle cell formation in endothelial cells.

Kaposi's sarcoma herpesvirus (KSHV) Fas-associated death domain (FADD)-like interleukin-1 beta-converting enzyme (FLICE)-inhibitory protein, vFLIP, has antiapoptotic properties, is a potent activator of the NF-κB pathway, and induces the formation of endothelial spindle cells, the hallmark of Kaposi's sarcoma, when overexpressed in primary endothelial cells. We used a reverse genetics approach to study several functions of KSHV vFLIP in the context of the whole viral genome. Deletion of the gene encoding vFLIP from a KSHV genome cloned in a bacterial artificial chromosome (BAC) reduced the ability of the virus to persist and induce spindle cell formation in primary human umbilical vein endothelial cells (HUVECs). Only a few, mainly interferon (IFN)-responsive, genes were expressed in wild-type KSHV (KSHV-wt)-infected endothelial cells at levels higher than those in KSHV-ΔFLIP-infected endothelial cells, in contrast to the plethora of cellular genes induced by overexpressed vFLIP. In keeping with this observation, vFLIP induces the phosphorylation of STAT1 and STAT2 in an NF-κB-dependent manner in endothelial cells. vFLIP-dependent phosphorylation of STAT1 and STAT2 could be demonstrated after endothelial cells were infected with KSHV-wt, KSHV-ΔFLIP, and a KSHV-vFLIP revertant virus. These findings document the impact of KSHV vFLIP on the transcriptome of primary endothelial cells during viral persistence and highlight the role of vFLIP in the activation of STAT1/STAT2 and STAT-responsive cellular genes by KSHV.

The contribution of systems biology and reverse genetics to the understanding of Kaposi's sarcoma-associated herpesvirus pathogenesis in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 is the causative agent of the endothelial cell-derived tumour Kaposi's sarcoma. Herpesviruses possess large complex genomes which provide many options to regulate cellular physiology during the viral life cycle and in the course of tumourigenicity. Novel techniques of systems biology and reverse genetics are increasingly applied to dissect the complex interaction of KSHV with endothelial cells. This review will outline novel results and pitfalls of these technologies in the elucidation of KSHV pathogenicity.

Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-kappaB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-kappaB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS.

Influence of HLA alleles on shedding of Kaposi sarcoma-associated herpesvirus in saliva in an African population.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) is endemic in sub-Saharan Africa. Infection in childhood involves mother-to-child transmission and transmission between siblings or other close contacts. Large amounts of viral DNA in saliva have been linked to transmission from mother to child. To investigate factors contributing to the shedding of KSHV in the saliva of mothers in rural South Africa, we sequenced the HLA-A alleles of 448 mothers and the HLA-DRB1 alleles of 363 mothers and compared their HLA types with viral loads in saliva. METHODS: Viral load was quantified with real-time polymerase chain reaction on DNA extracted from saliva. HLA-A and HLA-DRB1 allele groups were determined by sequencing-based typing. RESULTS: We found that 2 HLA-A alleles, A*6801 and A*4301, and 1 HLA-DRB1 allele group, DRB1*04, were associated with shedding of KSHV in saliva. KSHV could be detected more frequently in mothers carrying at least 1 copy of HLA-A*6801 or HLA-A*4301, and higher viral loads were found in HLA-A*68- and HLA-DRB1*04-carrying mothers. CONCLUSIONS: These findings could suggest that 2 HLA-A alleles, A*6801 and A*4301, and 1 HLA-DRB1 allele group, DRB1*04, that are more frequent in African populations might be associated with an impaired control of KSHV and, hence, increased shedding in saliva.

Mother-to-child transmission of human herpesvirus-8 in South Africa.

To investigate transmission of human herpesvirus (HHV)-8, 2546 mother-child pairs were recruited from rural clinics in South Africa and were tested for antibodies against lytic and latent HHV-8 antigens. The prevalence of antibodies in children increased with increasing maternal antibody titer (lytic, chi 21=26, and P<.001; latent, chi 21=55, and P<.001). HHV-8 DNA was detectable in 145 of 978 maternal saliva samples (mean virus load, 488,450 copies/mL; range, 1550-660,000 copies/mL) and in 12 of 43 breast-milk samples (mean virus load, 5800 copies/mL; range, 1550-12,540 copies/mL). The prevalence of HHV-8 DNA in maternal saliva was unrelated to latent anti-HHV-8 antibody status but was higher in mothers with the highest titers of lytic antibodies than in other mothers (34% vs. 8%; P<.001). The prevalence of lytic anti-HHV-8 antibodies in children was 13% (70/528) if the mother did not have HHV-8 in saliva and was 29% (8/28) if the mother had a high HHV-8 load (>50,000 copies/mL) in saliva (odds ratio, 2.6; 95% confidence interval, 1.1-6.2). The presence of HHV-8 DNA in maternal saliva was unrelated to latent antibodies in children. Saliva could be a route of transmission of HHV-8 from person to person, although other routes cannot be ruled out.