Evolutionary analysis of LMP-1 genetic diversity in EBV-associated nasopharyngeal carcinoma: Bioinformatic insights into oncogenic potential.

EBV latent membrane protein 1 (LMP-1) is an important oncogene involved in the induction and maintenance of EBV infection and the activation of several cell survival and proliferative pathways. The genetic diversity of LMP-1 has an important role in immunogenicity and tumorigenicity allowing escape from host cell immunity and more metastatic potential of LMP-1 variants. This study explored the evolutionary of LMP-1 in EBV-infected patients at an advanced stage of nasopharyngeal carcinoma (NPC). Detection of genetic variability in LMP-1 genes was carried out using Sanger sequencing. Bioinformatic analysis was conducted for translation and nucleotide alignment. Phylogenetic analysis was used to construct a Bayesian tree for a deeper understanding of the genetic relationships, evolutionary connections, and variations between sequences. Genetic characterization of LMP-1 in NPC patients revealed the detection of polymorphism in LMP-1 Sequences. Motifs were identified within three critical LMP-1 domains, such as PQQAT within CTAR1 and YYD within CTAR2. The presence of the JACK3 region at specific sites within CTAR3, as well as repeat regions at positions (122-132) and (133-143) within CTAR3, was also annotated. Additionally, several mutations were detected including 30 and 69 bp deletions, 33 bp repeats, and 15 bp insertion. Although LMP-1 strains appear to be genetically diverse, they are closely related to 3 reference strains: prototype B95.8, Med- 30 bp deletion, and Med + 30 bp deletion. In our study, one of the strains harboring the 30 bp deletion had both bone and bone marrow metastasis which could be attributed to the fact that LMP-1 is involved in tumor metastasis, evasion and migration of NPC cells. This study provided valuable insights into genetic variability in LMP-1 sequences of EBV in NPC patients. Further functional studies would provide a more comprehensive understanding of the molecular characteristics, epidemiology, and clinical implications of LMP-1 polymorphisms in EBV-related malignancies.

Prevalence and Genetic Diversity of Cross-Assembly Phages in Wastewater Treatment Plants in Riyadh, Saudi Arabia.

The most common DNA virus found in wastewaters globally is the cross-assembly phage (crAssphage). King Saud University wastewater treatment plant (KSU-WWTP); Manfoha wastewater treatment plant (MN-WWTP); and the Embassy wastewater treatment plant (EMB-WWTP) in Riyadh, Saudi Arabia were selected, and 36 untreated sewage water samples during the year 2022 were used in the current study. The meteorological impact on crAssphage prevalence was investigated. CrAssphage prevalence was recorded using PCR and Sanger sequencing. The molecular diversity of crAssphage sequences was studied for viral gene segments from the major capsid protein (MCP) and membrane protein containing the peptidoglycan-binding domain (MP-PBD). KSU-WWTP and EMB-WWTP showed a higher prevalence of crAssphage (83.3%) than MN-WWTP (75%). Phylogenetic analysis of MCP and MP-PBD segments depicted a close relationship to the Japanese isolates. The MCP gene from the current study's isolate WW/2M/SA/2022 depicted zero evolutionary divergence from 3057_98020, 2683_104905, and 4238_99953 isolates (d = 0.000) from Japan. A significant influence of temporal variations on the prevalence of crAssphage was detected in the three WWTPs. CrAssphage displayed the highest prevalence at high temperatures (33-44 °C), low relative humidity (6-14%), and moderate wind speed (16-21 Km/h). The findings provided pioneering insights into crAssphage prevalence and its genetic diversity in WWTPs in Riyadh, Saudi Arabia.

Molecular Epidemiology and Surveillance of Human Adenovirus and Rotavirus A Associated Gastroenteritis in Riyadh, Saudi Arabia.

In Saudi Arabia, acute gastroenteritis (GE) is a common illness affecting children and adults; however, the extent to which human rotavirus A (HRV) and human adenovirus (HAdV) strains contribute to the condition is unclear. The surveillance of the GE-causing viruses, HRV and HadV, was performed using polymerase chain reaction, sequencing, and phylogenetic analysis at King Khalid University Hospital. The associations between virus prevalence and meteorological factors were analyzed. The prevalence of HAdV was recorded (7%), followed by HRV (2%). On a gender basis, HAdV infections were found to be dominant in females (5:2) (U = 407.5; p < 0.0001), whereas HRV was only detected in males (U = 50; p < 0.0001). A significantly higher HAdV prevalence was recorded at the age of 3.5 ± 0.63 years (21.1%; p = 0.00047), whereas HRV cases were found equally distributed between <3 years and 3-5 years. The highest HAdV prevalence was recorded in autumn, followed by winter and spring. A significant correlation was detected between humidity and the total number of recorded cases (p = 0.011). Phylogenetic analysis depicted the dominance of HAdV type 41 and the G2 lineage of HRV among circulating strains. The current study uncovered the epidemiology and genotypes of HRV and HadV, and provided forecasting equations for monitoring climatic-mediated outbreaks.

Pepper Mild Mottle Virus as a Potential Indicator of Fecal Contamination in Influents of Wastewater Treatment Plants in Riyadh, Saudi Arabia.

Several indicators of fecal pollution in water resources are continuously monitored for their reliability and, of particular interest, their correlation to human enteric viruses-not justified by traditional bacterial indicators. Pepper mild mottle virus (PMMoV) has recently been proposed as a successful viral surrogate of human waterborne viruses; however, in Saudi Arabia there are no available data in terms of its prevalence and concentration in water bodies. The concentration of PMMoV in three different wastewater treatment plants (King Saud University (KSU), Manfoha (MN), and Embassy (EMB) wastewater treatment plants (WWTP)) was measured using qRT-PCR during a one-year period and compared to the human adenovirus (HAdV), which is highly persistent and considered an indicator for viral-mediated fecal contamination. PMMoV was found in ~94% of the entire wastewater samples (91.6-100%), with concentrations ranging from 62 to 3.5 × 107 genome copies/l (GC/l). However, HAdV was detected in 75% of raw water samples (~67-83%). The HAdV concentration ranged between 1.29 × 103 GC/L and 1.26 × 107 GC/L. Higher positive correlation between PMMoV and HAdV concentrations was detected at MN-WWTP (r = 0.6148) than at EMB-WWTP (r = 0.207). Despite the lack of PMMoV and HAdV seasonality, a higher positive correlation (r = 0.918) of PMMoV to HAdV was recorded at KSU-WWTP in comparison to EMB-WWTP (r = 0.6401) around the different seasons. Furthermore, meteorological factors showed no significant influence on PMMoV concentrations (p > 0.05), thus supporting the use of PMMoV as a possible fecal indicator of wastewater contamination and associated public health issues, particularly at MN-WWTP. However, a continuous monitoring of the PMMoV distribution pattern and concentration in other aquatic environments, as well as its correlation to other significant human enteric viruses, is essential for ensuring its reliability and reproducibility as a fecal pollution indicator.

Increased Prevalence of EBV Infection in Nasopharyngeal Carcinoma Patients: A Six-Year Cross-Sectional Study.

Epstein Barr Virus (EBV) is implicated in the carcinogenesis of nasopharyngeal carcinoma (NPC) and currently associated with at least 1% of global cancers. The differential prognosis analysis of NPC in EBV genotypes remains to be elucidated. Medical, radiological, pathological, and laboratory reports of 146 NPC patients were collected retrospectively over a 6-year period between 2015 and 2020. From the pathology archives, DNA was extracted from tumor blocks and used for EBV nuclear antigen 3C (EBNA-3C) genotyping by nested polymerase chain reaction (PCR). We found a high prevalence of 96% of EBV infection in NPC patients with a predominance of genotype I detected in 73% of NPC samples. Histopathological examination showed that most of the NPC patients were in the advanced stages of cancer: stage III (38.4%) or stage IV-B (37.7%). Only keratinized squamous cell carcinoma was significantly higher in EBV negative NPC patients compared with those who were EBV positive (OR = 0.01, 95%CI = (0.004-0.32; p = 0.009)), whereas the majority of patients (91.8%) had undifferentiated, non-keratinizing squamous cell carcinoma, followed by differentiated, non-keratinizing squamous cell carcinoma (7.5%). Although NPC had metastasized to 16% of other body sites, it was not associated with EBV infection, except for lung metastasis. A statistically significant reverse association was observed between EBV infection and lung metastasis (OR = 0.07, 95%CI = (0.01-0.51; p = 0.008)). Although 13% of NPC patients died, the overall survival (OS) mean time was 5.59 years. Given the high prevalence of EBV-associated NPC in our population, Saudi could be considered as an area with a high incidence of EBV-associated NPC with a predominance of EBV genotype I. A future multi-center study with a larger sample size is needed to assess the true burden of EBV-associated NPC in Saudi Arabia.

Novel findings in context of molecular diversity and abundance of bacteriophages in wastewater environments of Riyadh, Saudi Arabia.

The diversity among bacteriophages depends on different factors like ecology, temperature conditions and genetic pool. Current study focused on isolation, identification and diversity of phages from 34 sewage water samples collected from two different wastewater treatment plants (WWTPs), King Saud University wastewater treatment plants (KSU-WWTP) and Manfoha wastewater treatment plants (MN-WWTP) in Riyadh, Saudi Arabia. Samples were analyzed by PCR and Next Generation Sequencing (NGS). Siphoviridae, Podoviridae and Myoviridae families were detected by family-specific PCR and highest prevalence of Myoviridae 29.40% was found at MN-WWTP followed by 11.76% at KSU-WWTP. Siphoviridae was detected 11.76% at MN-WWTP and 5.88% at KSU-WWTP. Lowest prevalence for Podoviridae family (5.88%) was recorded at MN-WWTP. Significant influence of temporal variations on prevalence of Myoviridae and Siphoviridae was detected in both WWTP and MN-WWTP, respectively. Highest phage prevalence was obtained in August (75%), followed by September (50%). Highest phage prevalence was recorded at a temperature range of 29-33°C. Significant influence of temperature on the prevalence of Myoviridae phages was detected at MN-WWTP. Four bacteriophages with various abundance levels were identified by NGS. Cronobacter virus Esp2949-1 was found first time with highest abundance (4.41%) in wastewater of Riyadh. Bordetella virus BPP1 (4.14%), Dickeya virus Limestone (1.55%) and Ralstonia virus RSA1 (1.04%) were also detected from samples of MN-WWTP. Highest occurrence of Bordetella virus BPP1 (67%) and (33.33%) was recorded at KSU-WWTP and MN-WWTP, respectively. Highest Bordetella virus BPP1 occurrence was recorded in September (50%) followed by August (40%). The findings of study showed new insights of phage diversity from wastewater sources and further large-scale data studies are suggested for comprehensive understanding.

Distribution and Molecular Identification of Culex pipiens and Culex tritaeniorhynchus as Potential Vectors of Rift Valley Fever Virus in Jazan, Saudi Arabia.

Entomologic investigations were conducted in the Al-Darb, Al-Reath, Al-Aridah, Abuareesh, Al-Ahad, Samttah, Sabyah, Damad and Beash areas by CO2-baited CDC miniature light traps in the Jazan region. Vectors were identified morphologically, as well as COI gene segment amplification and sequencing. The relative abundance (RA%) and pattern of occurrence (C%) were recorded. The presence of the Rift Valley fever virus (RVFV) in pooled mosquito samples was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). Culex pipiens (C. pipiens) and Culex tritaeniorhynchus (C. tritaeniorhynchus) were found with RA% values of 96% and 4%, respectively, in the region. Significant variations in vector population densities were observed in different districts. The C. pipiens was found highly abundant in all districts and RA% value (100%) was recorded in the Al-Darb, Al-Reath, Al-Aridah, Samttah and Damad areas, whereas RA% values (93.75%, 93.33%, 92.30% and 91.66%) were noted in Al-Ahad, Sabyah, Abuareesh and Beash districts, respectively. RA% values for C. tritaeniorhynchus were recorded as 8.33%, 7.70%, 6.66% and 6.25% in Beash, Abuareesh, Sabyah and Al-Ahad areas, respectively. The pattern of occurrence for C. pipiens and C. tritaeniorhynchus was recorded as 100% and 44.4% in the region. Phylogenetic analysis of C. pipiens and C. tritaeniorhynchus exhibited a close relationship with mosquitoes from Kenya and Turkey, respectively. All mosquito samples tested by RT-PCR were found negative for RVFV. In summary, the current study assessed the composition, abundance, distribution of different mosquito vectors and presence of RVFV in different areas of the Jazan region. Our data will help risk assessments of RVFV future re-emergence in the region.

Evaluation of three different concentration and extraction methods for recovery efficiency of human adenovirus and human rotavirus virus A.

Routine wastewater treatment plants (WWTPs) effluents monitoring is essential because of enteric viruses' low infectious dose beyond molecular detectability. In current study methods for concentration and extraction, inter-method compatibility and recovery efficiency of spiked human adenovirus (HAdV) and human rotavirus A (RVA) were evaluated. For virus concentration, polyethylene glycol precipitation (PEG), charged membrane-based adsorption/elution (CMAE), and glass wool-based concentration (GW) methods were used. Nucleic acid was extracted by PowerViral™ Environmental RNA/DNA Isolation (POW), ZymoBIOMICS™ RNA extraction (ZYMO) and Wizard® Genomic DNA Purification (WGDP) and samples were analyzed by Real-Time PCR. CMAE method yielded significantly higher concentrations for both ARQ (Armored-RNA Quant) and RVA compared to PEG (P =  0.001 and 0.003) and GW (P <  0.0001). Highest HAdV concentration was obtained by PEG (P =  0.001 and < 0.0001) in relation to CMAE and GW. ZYMO yielded a significantly higher ARQ and RVA concentrations (P =  0.03 and 0.0057), whereas significantly higher concentration was obtained by POW for HAdV (P =  0.032). CMAE × ZYMO achieved the highest recovery efficiencies for ARQ (69.77 %) and RVA (64.25, respectively, while PEG × POW present efficiency of 9.7 % for HAdV. These findings provide guidance for understanding of method-related biases for viral recovery efficiency.

Novel insights of waterborne human rotavirus A in Riyadh (Saudi Arabia) involving G2 predominance and emergence of a thermotolerant sequence.

The routine evaluation of water environments is necessary to manage enteric virus-mediated fecal contamination and the possible emergence of novel variants. Here, we detected human rotavirus A (HRVA) circulating in two wastewater treatment plants, two lakes, irrigation water and a wastewater landfill located in Riyadh. VP7-derived surface protein sequences were assessed by phylogenetic analyses and inspection of thermotolerance-mediated secondary structure and seasonal variation. HRVA was most prevalent at An-Nazim wastewater landfill (AN-WWLF; 63.89%). Phylogenetic analyzes revealed the predominance of HRVA G2 lineage for the first time in Saudi Arabia. Moreover, a single HRVA sequence (2B64I-ANLF3/2018) was recovered at 45 °C from AN-WWLF; secondary structure prediction indicated that this sequence was thermotolerant with a high hydrophobicity, an absence of Ramachandran outliers, and a higher content of proline patches on the protein surface. Varied relationships were significantly observed between sampling areas influenced by temperature ranges (p < 0.05). HRVA prevalence was influenced by seasonal variations, favoring moderate temperatures in late autumn and early winter in all locations. However, a significant temperature impact was detected in Wadi-Hanifah Lake (p = 0.01). Our study extends the knowledge of currently circulating HRVA genotypes, and indicates the probable emergence of thermotolerant strains and seasonally mediated HRVA prevalence.

Human Adenovirus Molecular Characterization in Various Water Environments and Seasonal Impacts in Riyadh, Saudi Arabia.

The regular monitoring of water environments is essential for preventing waterborne virus-mediated contamination and mitigating health concerns. We aimed to detect human adenovirus (HAdV) in the Wadi Hanifah (WH) and Wadi Namar (WN) lakes, King Saud University wastewater treatment plant (KSU-WWTP), Manfouha-WWTP, irrigation water (IW), and AnNazim landfill (ANLF) in Riyadh, Saudi Arabia. HAdV hexon sequences were analyzed against 71 HAdV prototypes and investigated for seasonal influence. ANLF had the highest HAdV prevalence (83.3%). Remarkably, the F species of HAdV, especially serotype 41, predominated. Daily temperature ranges (22-45 °C and 10-33 °C) influenced the significance of the differences between the locations. The most significant relationship of ANLF and IW to WH and KSU-WWTP was found at the high-temperature range (p = 0.001). Meanwhile, WN was most correlated to ANLF at the low-temperature range (p < 0.0001). Seasonal influences on HAdV prevalence were insignificant despite HAdV's high prevalence in autumn and winter months, favoring low temperatures (high: 22-25 °C, low: 14-17 °C) at five out of six locations. Our study provides insightful information on HAdV prevalence and the circulating strains that can address the knowledge gap in the environmental impacts of viruses and help control viral diseases in public health management.

Molecular adaptive evolution of SARS-COV-2 spike protein in Saudi Arabia.

The sequences of SARS-CoV-2 spike (S) from Saudi Arabia along with SARS-CoV and bat SARS-like CoVs were obtained. Positive selection analysis and secondary structure investigation of spike sequences were performed. Adaptive molecular evolution was observed in SARS-CoV-2 displayed by positive selection pressure at N-terminal domain (NTD; codons 41, 163, 174 and 218), Receptor binding domain (RBD; codons 378 and 404) and S1/S2 Cleavage site (codon 690). Furthermore, the spike protein secondary structure depicted by the homo-trimer structure showed a high similarity between Saudi SARS-CoV-2 isolate and the parental strain (bat SL-COVZC45). Despite the high similarity depicted in the spike sequence model alignment, it displayed a significant difference when each chain was treated solely owing to 7 motif differences in the three composing chains. In addition, SARS-CoV-2 S trimer model uncovered the presence of N-acetyl glucosamine ligands. Eventually, 3C-like proteinase cleavage site was observed in S2 domain could be used as a site for drug discovery. Genetics and molecular evolutionary facts are useful for assessment of evolution, host adaptation and epidemic patterns ultimately helpful for adaptation of control strategies.

A pandemic risk assessment of middle east respiratory syndrome coronavirus (MERS-CoV) in Saudi Arabia.

Since the initial emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, a high incidence rate has been observed in Saudi Arabia. This suggests that the country is at continuous risk. The epidemic level of MERS-CoV infection was examined in Saudi Arabia by the Susceptible-Infectious-Recovered (SIR) model using a Bayesian approach for estimation of time dependent reproduction number (R) across a two-year interval (May, 2013-May, 2015) in five defined clusters, followed by sensitivity analysis of the most significant clusters. Significant MERS-CoV peaks were detected in the period between March and May of each year. Moreover, MERS-CoV infection was highlighted in western (40.8%) and central (31.9%) regions, followed by eastern region (20%). The temporal-based Bayesian approach indicated a sub-critical epidemic in all regions in the baseline scenario (R: 0.85-0.97). However, R potential limit was exceeded in the sensitivity analysis scenario in only central and western regions (R: 1.08-1.12) that denoted epidemic level in those regions. The impact of sporadic cases was found relatively insignificant and pinpointed to the lack of zoonotic influence on MERS-CoV transmission dynamics. The results of current study would be helpful for evaluation of future progression of MERS-CoV infections, better understanding and control interventions.

Respiratory Tract Viral Infections and Coinfections Identified by Anyplex™ II RV16 Detection Kit in Pediatric Patients at a Riyadh Tertiary Care Hospital.

Respiratory infections are caused by an array of viruses, and limited information is available about viral coexistence, comparative symptoms, and the burden of illness. This retrospective cohort study aimed to determine the etiological agents responsible for respiratory tract infections by Anyplex II RV16 detection kit (RV16, Seegene), involving 2266 pediatric patients with respiratory infections admitted to the Department of Pediatrics at King Abdul-Aziz Medical City, Ministry of National Guard, Riyadh, from July 2014 to June 2015. The most frequent respiratory infections were recorded in the 1 to 5 year age group (44.7%). Rhinovirus (32.5%), Adenovirus (16.9%), and Respiratory syncytial virus (RSV) B (10.4%) were most common. In single viral infections, Rhinovirus (41.2%), Metapneumovirus (15.3%), and Bocavirus (13.7%) were most frequent. In multiple viral infections, Rhinovirus (36.7%), Adenovirus (35.2%), Bocavirus (11.2), RSV B (7.8%), and RSV A (6.7%) were most frequent. No significant difference was observed in clinical presentations; however, rhinorrhea and hypodynamia were significantly associated with viral respiratory infections. Most respiratory viral pathogens peaked during December, January, March, and April. Rhinovirus, Adenovirus, and Bocavirus circulations were detected throughout the year. Winter peaks were recorded for Rhinovirus, RSV B, Adenovirus, and RSV A, whereas the Metapneumovirus, and the Bocavirus peaked in March and April. These findings enhance understanding of viral etiology and distribution to improve respiratory infection management and treatment.

Non-structural proteins of arthropod-borne bunyaviruses: roles and functions.

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod.

Mutational analysis of the Bunyamwera orthobunyavirus nucleocapsid protein gene.

The bunyavirus nucleocapsid protein, N, is a multifunctional protein that encapsidates each of the three negative-sense genome segments to form ribonucleoprotein complexes that are the functional templates for viral transcription and replication. In addition, N protein molecules interact with themselves to form oligomers, with the viral L (RNA polymerase) protein, with the carboxy-terminal regions of either or both of the virion glycoproteins, and probably also with host cell proteins. Bunyamwera virus (BUNV), the prototype bunyavirus, encodes an N protein of 233 amino acids in length. To learn more about the roles of individual amino acids in the different interactions of N, we performed a wide-scale mutagenic analysis of the protein, and 110 single-point mutants were obtained. When the mutants were employed in a minireplicon assay to examine their effects on viral RNA synthesis, a wide range of activities compared to those of wild-type N protein were observed; changes at nine amino acid positions resulted in severely impaired RNA synthesis. Seventy-seven mutant clones were selected for use in the bunyavirus reverse genetics system, and 57 viable recombinant viruses were recovered. The recombinant viruses displayed a range of plaque sizes and titers in cell culture (from approximately 10(3) to 10(8) PFU/ml), and a number of viruses were shown to be temperature sensitive. Different assays were applied to determine why 20 mutant N proteins could not be recovered into infectious virus. Based on these results, a preliminary domain map of the BUNV N protein is proposed.