Human bocavirus in Saudi Arabia: Molecular epidemiology and Co-infections among children with acute respiratory tract infections during 2014-2016.

Respiratory tract infections due to a variety of viruses continue to threaten the human population worldwide, particularly in developing countries. Among the responsible viruses, Human Bocavirus (HBoV), a novel discovered virus, causes respiratory tract and gastroenteritis disorders in young children. In Saudi Arabia, data regarding virus molecular epidemiology and evolution and its implication in respiratory tract infection are scarce. In the current study, genetic diversity and circulation pattern of HBoV-1 among hospitalized children due to acute respiratory tract infection (ARTI) during two consecutive years were charted. We found that 3.44% (2014/2015) and 11.25% (2015/2016) of children hospitalized due to ARTI were infected by HBoV-1. We have shown that HBoV was detected year-round without a marked seasonal peak. HBoV-1 also was co-detected with one or multiple other respiratory viruses. The multisequence analysis showed high sequence identity (∼99%) (few point mutation sites) between strains of each genotype and high sequence variation (∼79%) between HBoV-1 and the other 3 genotypes. Phylogenetic analysis showed the clustering of the study's isolates in the HBoV-1 subclade. Our data reveal that genetically conserved HBoV-1 was circulating among admitted children during the course of the study. Further epidemiological and molecular characterization of multiple HBoV-1 strains for different years and from all regions of Saudi Arabia are required to understand and monitor the virus evolution.

Chemical Composition, Antioxidant, Anticancer, and Antibacterial Activities of Roots and Seeds of Ammi visnaga L. Methanol Extract.

For centuries, plants and their components have been harnessed for therapeutic purposes, with Ammi visnaga L. (Khella) being no exception to this rich tradition. While existing studies have shed light on the cytotoxic and antimicrobial properties of seed extracts, there remains a noticeable gap in research about the antimicrobial, antioxidant, and anticancer potential of root extracts. This study seeks to address this gap by systematically examining methanol extracts derived from the roots of A. visnaga L. and comparing their effects with those of seed extracts specifically against breast cancer cells. Notably, absent from previous investigations, this study focuses on the comparative analysis of the antimicrobial, antioxidant, and anticancer activities of both root and seed extracts. The methanol extract obtained from A. visnaga L. seeds demonstrated a notably higher level of total phenolic content (TPC) than its root counterpart, measuring 366.57 ± 2.86 and 270.78 ± 2.86 mg GAE/g dry weight of the dry extract, respectively. In the evaluation of antioxidant activities using the DPPH method, the IC50 values for root and seed extracts were determined to be 193.46 ± 17.13 µg/mL and 227.19 ± 1.48 µg/mL, respectively. Turning our attention to cytotoxicity against breast cancer cells (MCF-7 and MDA-MB-231), both root and seed extracts displayed similar cytotoxic activities, with IC50 values of 92.45 ± 2.14 µg/mL and 75.43 ± 2.32 µg/mL, respectively. Furthermore, both root and seed extracts exhibited a noteworthy modulation of gene expression, upregulating the expression of caspase and Bax mRNA levels while concurrently suppressing the expression of anti-apoptotic genes (Bcl-xL and Bcl-2), thereby reinforcing their potential as anticancer agents. A. visnaga L. seed extract outperforms the root extract in antimicrobial activities, exhibiting lower minimum inhibitory concentrations (MICs) of 3.81 ± 0.24 to 125 ± 7.63 µg/mL. This highlights the seeds' potential as potent antibacterial agents, expanding their role in disease prevention. Overall, this study underscores the diverse therapeutic potentials of A. visnaga L. roots and seeds, contributing to the understanding of plant-derived extracts in mitigating disease risks.

Genetic analysis of human parainfluenza type 2 virus in Riyadh, Saudi Arabia.

The extensive mass gathering of pilgrims from all over the world, as well as the constant flow of foreign workers via country entry crossings, raises the likelihood of respiratory virus outbreaks spreading and evolving in Saudi Arabia. Here, we report the sequence and phylogenetic analysis of the human parainfluenza type-2 (HPIV-2) in nasopharyngeal aspirates (NPAs) collected from Riyadh, Saudi Arabia, from 2020/21 to 2021/22 seasons. RNA was extracted from the clinical samples and subjected to RT-PCR analysis for the detection of IAV and IBV. The full-length HN gene of HPIV-2 was amplified and sequenced. Multiple sequence alignments (both nucleotides and deduced amino acids) were aligned using Clustal W, MegAlign program of Lasergene software, and MEGA 7.0. HPIV-2 was found in (4; 2% of 200) NPAs. Sequence and phylogenetic analysis results showed that indicated a genotype shifting from G3 to G4a with 83% sequence homology 62-M786 from Japan, which was prominent throughout the winter seasons of 2008/09. Multiple amino acid sequence alignment revealed 25 sites of possible difference between G3 genotypes and G4a. A total of twenty- two of these locations were shared by the other G4a genotypes, whereas three positions, 67 V, 175 S, and 377Q, were exclusively shared by G3. Only eight conserved N-glycosylation sites were found at amino acids 6(NLS), 286(NTT), 335(NIT), 388(NNS), 498(NES), 504(NPT), 517(NTT), and 539(NGT) in four Riyadh isolates. Our findings also revealed that the G4a genotype of HPIV-2 predominated in our samples population during the winter seasons of 2020/21 and 2021/22. Further research with a larger sample size covering numerous regions of Saudi Arabia throughout different epidemic seasons is needed to achieve an improved knowledge of HPIV-2 circulation.

Levels and profiles of pentabromodiphenyl ether contaminants in human breast milk from Riyadh, Saudi Arabia.

OBJECTIVES: To investigate levels of pentabromodiphenyl ether (PBDEs) in breast milk samples from healthy mothers who had lived in Riyadh for the last 5 years. METHODS: In this cross sectional study, 75 samples were collected and were extracted, cleaned by solid-phase extraction (SPE) and PBDEs analysis was done using gas chromatography-mass spectrometry. RESULTS: Total PBDEs (∑PBDEs) ranged from 0.2 to 3.6 ng/g lipid weight (lw). BDE-47, -153, -99, and -209 were the dominant congeners. The mothers in this study consumed more meat (69%), followed by the egg (50%), and milk (36%). The majority of donors consumed fish (44%) and egg (33%) 2 times per week. The majority of the participating mothers had completed higher education (68%). All PBDE congeners were detected in the human breast milk samples with high detection frequency (98%). The dominant congener was BDE 47, accounting more than 39% of all BDE congeners, followed by BDE-99 and BDEs 153 which accounted for18% and 12% of the total BDE congeners respectively. CONCLUSION: Higher rates of meat and poultry consumption were positively associated with higher breast milk levels of ∑PBDEs. The significant levels of PBDEs that occur in the meat and poultry reared in Saudi Arabia need further investigation especially as Saudis among largest consumers of poultry meat.

Molecular Profiling of Inflammatory Mediators in Human Respiratory Syncytial Virus and Human Bocavirus Infection.

Infections due to human respiratory syncytial virus (HRSV) and human bocavirus (HBoV) can mediate the release of several pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, which are usually associated with disease severity in children. In this study, the change in the expression profile of cytokines and chemokines were determined during HRSV, HBoV, and HRSV coinfection with HBoV in 75 nasopharyngeal aspirates (NPAs) samples, positive real-time reverse transcriptase PCR Assay (rRT-PCR) for HRSV (n = 36), HBoV (n = 23) infection alone or HRSV coinfection with HBoV (n = 16). The samples were collected from hospitalized children. qPCR-based detection revealed that the levels of IL-6, IL-8, IL-10, IL-13, IL-33, and G-CSF were significantly (p < 0.05) greater in patients than in controls. IL-4, IL-17, GM-CSF, and CCL-5 were significantly elevated in children with HRSV coinfection with HBoV than in other groups (p < 0.05). TNF-α, IL-6, IL-8, IL-10, IL-13, and IL-33 in children with HRSV were significantly increased in severe infections compared to mild infections. Whereas, IL-10, IL-13, and IL-33 were significantly increased in severe infection in compared a mild infection in children with HBoV. Further large-scale investigations involving isolates are needed to enhance our knowledge of the association between viral infections and cytokine expression patterns during the different stages of HRSV and HBoV infection.

Genetic Diversity and Evolutionary Kinetics of Influenza A Virus H3N2 Subtypes Circulating in Riyadh, Saudi Arabia.

Presence of a large foreign workforce and the annual gathering of people for pilgrimage from around the globe have significantly contributed to the emergence and diversity of respiratory viruses in Saudi Arabia. Here, we report the sequence and phylogenetic analysis of the H3N2 subtype of influenza A virus (IAV) in clinical samples collected from Riyadh, Saudi Arabia. Based on RT-PCR, IAV was found in 88 (28.3%) of the 311 samples screened. Of the 88-IAV positive samples, 43 (48.8%) were H1N1 subtype while the remaining 45 (51.2%) were found to be of the H3N2 subtype. Complete sequencing of HA and NA genes of H3N2 revealed, twelve and nine amino acid (AA) substitutions respectively, and importantly, these variations are absent in the current vaccine strains. Based on the phylogenetic analysis, the majority of H3N2 strains were grouped in the same clades as the vaccine strains. Importantly, the N-glycosylation sites at AA 135(NSS) were found to be unique to 6 strains in the investigated HA1 protein and were absent in the current vaccine strains. These data may have significant clinical implications in designing novel and population-based vaccines for IAV and underscore the need for regular monitoring of efficacy of vaccines due to emerging variants.

Prevalence of Human Coronaviruses in Children and Phylogenetic Analysis of HCoV-OC43 during 2016-2022 in Riyadh, Saudi Arabia.

With the emergence of SARS-CoV-2, routine surveillance combined with sequence and phylogenetic analysis of coronaviruses is urgently required. In the current study, the four common human coronaviruses (HCoVs), OC43, NL63, HKU1, and 229E, were screened in 361 clinical samples collected from hospitalized children with respiratory symptoms during four winter seasons. RT-PCR-based detection and typing revealed different prevalence rates of HCoVs across the four seasons. Interestingly, none of the four HCoVs were detected in the samples (n = 100) collected during the winter season of the COVID-19 pandemic. HCoV-OC43 (4.15%) was the most frequently detected, followed by 229E (1.1%). Partial sequences of S and N genes of OC43 from the winter seasons of 2015/2016 and 2021/2022 were used for sequence and phylogenetic analysis. Multiple sequence alignment of the two Saudi OC43s strains with international strains revealed the presence of sequence deletions and several mutations, of which some changed their corresponding amino acids. Glycosylation profiles revealed a number of O-and N-glycosylation sites in both genes. Based on phylogenetic analysis, four genotypes were observed with Riyadh strains grouped into the genotype C. Further long-term surveillance with a large number of clinical samples and sequences is necessary to resolve the circulation patterns and evolutionary kinetics of OC43 in Saudi Arabia.

ON-1 and BA-IX Are the Dominant Sub-Genotypes of Human Orthopneumovirus A&B in Riyadh, Saudi Arabia.

Human orthopneumovirus (HOPV) is the major viral pathogen responsible for lower respiratory tract infections (LRTIs) in infants and young children in Riyadh, Saudi Arabia. Yet, predominant HOPV subtypes circulating in this region and their molecular and epidemiological characteristics are not fully ascertained. A total of 300 clinical samples involving nasopharyngeal aspirates (NPAs), throat swabs, and sputum were collected during winter seasons of 2019/2020 and 2021/2022 for HOPV subtyping and genotyping. Of the 300 samples, HOPV was identified in 55 samples (18.3%) with a distinct predominance of type A viruses (81.8%) compared to type B viruses (18.2%). Importantly, the ON1 strain of HOPV-A and BA-IX strain of HOPV-B groups were found to be responsible for all the infections. Sequence analysis revealed a duplication region within 2nd HVR of G protein gene of ON1 and BA-IX strains. This nucleotide duplication exerted a profound effect on protein length and affinity towards cell receptors. Further, these modifications may aid the HOPV in immune evasion and recurrent infections. Data from this study showed that ON-1 genotype of HOPV-A and BA-IX genotype of HOPV-B were dominant in Riyadh, Saudi Arabia. Further, a duplication of sequence within 2nd HVR of G protein gene was found.

Development and Validation of Rapid In-House Diagnostic ELISA Kits for Detection of Human Orthopneumovirus in Clinical Samples.

Currently, the standard assay employed to diagnose human orthopneumovirus infection is real-time reverse transcriptase PCR assay (rRT-PCR), a costly and time-consuming procedure that requires the manipulation of infectious viruses. In addition to RT-PCR, serological tests can complement the molecular diagnostic methods and have proven to be important tools in sero-surveillance. In this study, we report the development, optimization, and validation of a novel and rapid in-house diagnostic ELISA kit to detect human orthopneumovirus in clinical samples. We developed three sensitive ELISA formats through the immunization of rats with novel recombinant pPOE-F or pPOE-TF vectors. The two vectors expressed either the full-length (pPOE-F) or the truncated form (pPOE-TF) of the fusion (F) protein. The developed ELISA kits were optimized for coating buffer, capture antibody, blocking buffer, sample antigen, detection antibodies, and peroxidase-conjugated antibody, and validated using 75 rRT-PCR-confirmed nasopharyngeal aspirate (NPA) human orthopneumovirus samples and 25 negative samples collected from hospitalized children during different epidemic seasons between 2014 and 2017. Our results indicate that rats immunized with pPOE-F or pPOE-TF showed significant induction of high levels of MPAs. Validation of the ELISA method was compared to the rRT-PCR and the sensitivity hierarchy of these developed ELISA assays was considered from highest to lowest: indirect competitive inhibition ELISA (93.3%) > indirect antigen-capture ELISA (90.6%) > direct antigen-capture ELISA (86.6%). The development of the rapid in-house diagnostic ELISA kits described in this study demonstrates that a specific, rapid and sensitive test for human orthopneumovirus antigens could be successfully applied to samples collected from hospitalized children during different epidemics and can help in the efficient diagnosis of respiratory syncytial viral infections.

SARS-CoV-2: An Overview of Virus Genetics, Transmission, and Immunopathogenesis.

The human population is currently facing the third and possibly the worst pandemic caused by human coronaviruses (CoVs). The virus was first reported in Wuhan, China, on 31 December 2019 and spread within a short time to almost all countries of the world. Genome analysis of the early virus isolates has revealed high similarity with SARS-CoV and hence the new virus was officially named SARS-CoV-2. Since CoVs have the largest genome among all RNA viruses, they can adapt to many point mutation and recombination events; particularly in the spike gene, which enable these viruses to rapidly change and evolve in nature. CoVs are known to cross the species boundaries by using different cellular receptors. Both animal reservoir and intermediate host for SARS-CoV-2 are still unresolved and necessitate further investigation. In the current review, different aspects of SARS-CoV-2 biology and pathogenicity are discussed, including virus genetics and evolution, spike protein and its role in evolution and adaptation to novel hosts, and virus transmission and persistence in nature. In addition, the immune response developed during SARS-CoV-2 infection is demonstrated with special reference to the interplay between immune cells and their role in disease progression. We believe that the SARS-CoV-2 outbreak will not be the last and spillover of CoVs from bats will continue. Therefore, establishing intervention approaches to reduce the likelihood of future CoVs spillover from natural reservoirs is a priority.

The emergence of subgenotype ON-1 of Human orthopneumovirus type A in Riyadh, Saudi Arabia: A new episode of the virus epidemiological dynamic.

Lower respiratory tract infections caused by Human orthopneumovirus are still a threat to the pediatric population worldwide. To date, the molecular epidemiology of the virus in Saudi Arabia has not been adequately charted. In this study, a total of 205 nasopharyngeal aspirate samples were collected from hospitalized children with lower respiratory tract symptoms during the winter seasons of 2014/15 and 2015/16. Human orthopneumovirus was detected in 89 (43.4%) samples, of which 56 (27.3%) were positive for type A and 33 (16.1%) were positive for type B viruses. The fragment that spans the two hypervariable regions (HVR1 and HVR2) of the G gene of Human orthopneumovirus A was amplified and sequenced. Sequence and phylogenetic analyses have revealed a genotype shift from NA1 to ON-1, which was prevalent during the winter seasons of 2007/08 and 2008/09. Based on the intergenotypic p-distance values, ON-1 was reclassified as a subgenotype of the most predominant genotype GA2. Three conserved N-glycosylation sites were observed in the HVR2 of Saudi ON-1 strains. The presence of a 23 amino acid duplicated region in ON-1 strains resulted in a higher number of O-glycosylation sites as compared to other genotypes. The data presented in this report outlined the replacement of NA1 and NA2 subgenotypes in Saudi Arabia with ON-1 within 7 to 8 years. The continuous evolution of Human orthopneumovirus through point mutations and nucleotide duplication may explain its ability to cause recurrent infections.