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Foot-and-mouth disease (FMD) is a serious highly contagious viral disease affecting all cloven-hoofed animals, and outbreaks can have a severe economic impact. An inactivated heptavalent oil-adjuvanted FMD vaccine (Aphtovac-7, MEVAC) was prepared from the foot-and-mouth disease virus (FMDV) strains A-Iran05, A-Africa-IV, O-PanAsia2, O-Manisa, O-EA3, SAT-2 Gharbia, and SAT-2 LIB-12. The vaccine potency and effectiveness were evaluated in three groups of 6- to 8-month-old calves and 200 adult dairy cattle under field conditions. All animals were vaccinated with the vaccine preparation, and the three groups of calves were challenged after 28 days by intradermolingual inoculation with 104 50% tissue culture infective dose (TCID50) of FMDV serotype A, O, or SAT-2. Mock-vaccinated calves (two per group) served as unvaccinated controls during the challenge test. Adult dairy cattle were tested for seroconversion using a virus neutralization test at 30, 60, and 120 days post-vaccination. All calves displayed complete protection against challenge with the different serotypes of FMDV when compared to the control groups. Serum samples collected after the primary and booster immunizations at 30 days post-vaccination contained high titers of protective antibodies (≥ 1/32; i.e. 1.5 log10). Antibodies persisted until the end of the study period (120 days), with a peak value around 60 days post-vaccination. The heptavalent FMD vaccine preparation was found to be potent and capable of providing a protective immune response under both experimental and field conditions.

Introduction: Gallid alphaherpesvirus 2 (GaHV-2) is a highly contagious oncogenic virus that causes Marek’s disease in chickens and occasionally in turkeys. Among 100 genes identified in GaHV-2 genome, the Meq gene appears to involve viral virulence, oncogenicity, and genetic diversity. Despite the use of Meq gene sequences in phylogenetic classification of GaHV-2 strains circulating in many countries worldwide, no integrated system exists yet. Methods: Turkeys from 2 commercial Egyptian farms were presented with signs of dullness, dehydration, and emaciation. Samples prepared from the internal organs were examined by histopathology and immunohistochemistry. Pools of the internal organs were analyzed by PCR for identification of GaHV-2, avian leucosis virus, and reticuloendotheliosis virus. The Meq gene of an Egyptian strain was sequenced and analyzed in comparison to 40 reference strains for generation of a universal system for phylogenetic classification of GaHV-2 strains. Results: Gross and histopathological examination revealed grayish-white soft masses in the internal organs characterized by diffuse infiltration of pleomorphic neoplastic cells. All lymphoma cells were identified as T-lymphocytes of CD3+ phenotype. Samples of both farms were only positive for GaHV-2 by PCR. Sequence analysis of the Meq gene has classified the current turkey strain as related to the Egyptian strains identified in chicken in 2012. A universal phylogenetic system for classification of GaHV-2 strains into 4 clusters was proposed. The vaccine strains were all grouped in cluster 2, and most of the classical American strains belonged to cluster 4. Cluster 1 was further divided into 3 subclusters (1.1–1.3). Conclusion: GaHV-2 was identified in turkeys for the first time in Africa and the Middle East. Sequence analysis of the Meq gene of the Egyptian strain along with a wide array of the global strains has enabled the construction of a novel phylogenetic classification system.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Acute lower respiratory tract infection is a major health problem that affects more than 15% of the total population of Saudi Arabia each year. Epidemiological studies conducted over the last three decades have indicated that viruses are responsible for the majority of these infections. The epidemiology of respiratory viruses in Saudi Arabia is proposed to be affected mainly by the presence and mobility of large numbers of foreign workers and the gathering of millions of Muslims in Mecca during the Hajj and Umrah seasons. Knowledge concerning the epidemiology, circulation pattern, and evolutionary kinetics of respiratory viruses in Saudi Arabia are scant, with the available literature being inconsistent. This review summarizes the available data on the epidemiology and evolution of respiratory viruses. The demographic features associated with Middle East respiratory syndrome-related coronavirus infections are specifically analyzed for a better understanding of the epidemiology of this virus. The data support the view that continuous entry and exit of pilgrims and foreign workers with different ethnicities and socioeconomic backgrounds in Saudi Arabia is the most likely vehicle for global dissemination of respiratory viruses and for the emergence of new viruses (or virus variants) capable of greater dissemination.

Hybridomas that secreted antibodies against aflatoxin B1 for multiple uses were prepared using a unique immunization schedule. Aflatoxin B1-BSA conjugate was used for immunization of Balb/c mice. Spleen cells were harvested from the hyper immunized mice to be fused with myeloma cell line (P3NS1) using polyethylene glycol 3000, 50% concentration as a fusogenic agent. The produced hybridomas were selected using HAT selective medium that was replaced by complete HT medium. From the 10thday after fusion, wells that contain colonies of hybridomas covering 30% or greater of the wells surface were screened for production of monoclonal antibodies against aflatoxin B1 using ELISA. 21 hybridomas were found to be reactive to aflatoxin B1. All were found to belong to IgG2aisotype except one was found to belong to IgM isotype. The prepared monoclonal antibodies and their application to immunoassays represents a useful and rapid quantitative measurement with high affinity and low detection limits in order to purify environmentally occurring levels of this carcinogen specially in areas at high risk for liver cancer.

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.

Background: COVID-19 has emerged as the most serious pandemic in the 21st century to date. COVID-19 patients may develop various disease symptoms that hinder the accurate clinical diagnosis. Summary: Routine diagnosis of COVID-19 requires complementary investigations, including computed tomography, immunological assays, and molecular assays like real-time RT-PCR, loop-mediated isothermal amplification, metagenomic next-generation sequencing, and clusters of regularly interspaced short palindromic repeats-based assays. Clinically approved antiviral drugs available for the COVID-19 treatment are very limited. The most common measurements that enhance health condition and patients’ viability are conservation fluid management, oxygen therapy, and antibiotics. Several therapeutic options have been developed or repurposed to prevent virus replication and/or modulate the immune response against virus infection. These options include various drugs that affect virus entry and membrane fusion, inhibit polymerase and protease activity, suppress the host pro-inflammatory cytokines, and utilize cell therapy approaches. Key Messages: In this review, we aimed to provide an up-to-date discussion on the current diagnostic options and therapeutic strategies used to control and manage COVID-19 in clinical and point-of-care settings.

Background The Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV) continues to exist in the Middle East sporadically. Thorough investigations of the evolution of human coronaviruses (HCoVs) are urgently required. In the current study, we studied amplified fragments of ORF1a/b, Spike (S) gene, ORF3/4a, and ORF4b of four human MERS-CoV strains for tracking the evolution of MERS-CoV over time. Methods RNA isolated from nasopharyngeal aspirate, sputum, and tracheal swabs/aspirates from hospitalized patients with suspected MERS-CoV infection were analyzed for amplification of nine variable genomic fragments. Sequence comparisons were done using different bioinformatics tools available. Results Several mutations were identified in ORF1a/b, ORF3/4a and ORF4b, with the highest mutation rates in the S gene. Five codons; 4 in ORF1a and 1 in the S gene, were found to be under selective pressure. Characteristic amino acid changes, potentially hosted and year specific were defined across the S protein and in the receptor-binding domain Phylogenetic analysis using S gene sequence revealed clustering of MERS-CoV strains into three main clades, A, B and C with subdivision of with clade B into B1 to B4. Conclusions In conclusion, MERS-CoV appears to continuously evolve. It is recommended that the molecular and pathobiological characteristics of future MERS-CoV strains should be analyzed on regular basis to prevent potential future outbreaks at early phases.

Nowadays, the human population is facing the third and may be the worst pandemic caused by human coronaviruses (CoVs). The virus was first reported in Wuhan, China on 31 December 2019 and spread within 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 many point mutation and recombination events; particularly in spike gene, that enable these viruses to rapidly change and evolve in nature. CoVs are known to cross the species boundaries by using different cellular receptors. SARS-CoV-2 is believed to originate in bats and transmitted to human being through an ill-defined intermediate host. 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 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 the natural reservoirs is a priority.

Background During the last few years since HBoV discovery, the virus has attracted less attention and appeared to be one of the most common acute respiratory tract pathogens in children. However, few studies focused on genetic characterization and epidemiology of HBoV in Saudi Arabia, leading us to evaluate the molecular epidemiology of HBoV strains among children with ARI. This study was designed to study the epidemiological, genetic diversity and circulation pattern of HBoV among hospitalized children (< 7 years) due to acute respiratory tract infection (ARTI) during two consecutive years. Nasopharyngeal aspirate (NPA) samples (n=196) were collected. HBoV and co-infection with other respiratory pathogens were identified using qRT-PCR. Positive samples were subsequently subjected to amplify full-length of VP1/VP2 gene, followed by sequencing and phylogenetic analyses. Results Our data showed that 3.4% (2014/2015) and 11.3% (2015/2016) of children hospitalized due to acute respiratory infections (ARI) were infected by HBoV-1. We have shown that HBoV-1 was detected year-round. HBoV-1 also was co-detected with one or multiple other respiratory viruses. Phylogenetic analysis of complete HBoV VP1/2 sequences showed no nucleotide variation and the circulation of HBoV-1. The multisequence analysis showed complete identity between HBoV isolated from 2014/2015 and 2015/2016, suggesting no genetic variability among the local circulating HBoV-1 genotypes. Our data reveal that genetically conserved HBoV-1 is circulating in Saudi Arabia. Conclusions The multisequence analysis showed complete identity between HBoV-1 isolated from 2014/2015 and 2015/2016, suggesting no genetic variability among the local circulating HBoV-1 genotypes. Our data reveal that genetically conserved HBoV-1 is circulating year-round in Saudi Arabia.

Coronaviruses (CoVs) produce a wide spectrum of disease syndromes in different mammalian and avian host species. These viruses are well-recognized for their ability to change tissue tropism, to hurdle the interspecies barriers and to adapt ecological variations. It is predicted that the inherent genetic diversity of CoVs caused by accumulation of point mutations and high frequency of homologous recombination is the principal determinant of these competences. Several CoVs (e.g. Severe acute respiratory syndrome-CoV, Middle East respiratory syndrome-CoV) have been recorded to cross the interspecies barrier, inducing different disease conditions in variable animal hosts. Bovine CoV (BCoV) is a primary cause of gastroenteritis and respiratory disease in cattle calves, winter dysentery in lactating cows and shipping fever pneumonia in feedlot cattle. Although it has long been known as a restrictive cattle pathogen, CoVs that are closely related to BCoV have been recognized in dogs, humans and in other ruminant species. Biologic, antigenic and genetic analyses of the so-called ‘bovine-like CoVs’ proposed classification of these viruses as host-range variants rather than distinct virus species. In this review, the different bovine-like CoVs that have been identified in domesticated ruminants (water buffalo, sheep, goat, dromedary camel, llama and alpaca) and wild ruminants (deer, wild cattle, antelopes, giraffes and wild goats) are discussed in terms of epidemiology, transmission and virus characteristics. The presented data denote the importance of these viruses in the persistence of BCoV in nature, spread to new geographical zones, and continuous emergence of disease epidemics in cattle farms.

The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codon-optimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M282–90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8+ T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses in the poultry industry worldwide. In this study, ILT outbreaks were reported on 30 farms located in eight Egyptian governorates between January 2018 and May 2019. Gross examination of diseased chickens revealed congestion and hemorrhage of laryngeal and tracheal mucosa with fibrinohemorrhagic casts and/or caseous material in the lumens. Histopathological examination showed epithelial sloughing, syncytium formation, heterophilic exudation, and development of eosinophilic intranuclear inclusion bodies. Infectious laryngotracheitis virus (ILTV) antigen was detected in the tracheal epithelium, infiltrated inflammatory cells, and syncytial cells, using immunohistochemistry. PCR targeting a portion of the thymidine kinase gene was further utilized to confirm the presence of ILTV DNA. The complete coding sequences of three envelope glycoprotein genes, gG, gD, and gJ, and a partial sequence of the infected cell polypeptide 4 (ICP4) gene from samples representing all of the farms and disease outbreaks were determined. Five prototype strains with unique sequences were chosen for detailed molecular characterization. Sequence comparisons and phylogenetic analysis of the partial ICP4 gene revealed that two strains were chicken embryo origin (CEO)-vaccine-like strains, and three were tissue culture origin (TCO)-vaccine-like strains. Analysis of the gJ gene sequence indicated that all of the strains were CEO vaccine-like strains. It was predicted that the latter three strains were recombinants of CEO- and TCO-vaccine-like strains. In conclusion, immunohistochemistry coupled with multi-genomic PCR sequencing proved to be efficient for identification and typing of ILTV strains during disease outbreaks. Both CEO-vaccine-like and recombinant virus strains were circulating in Egypt during the 2018 and 2019 outbreaks.

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.

Background/Aims: Hepatitis B virus (HBV) continues to be one of the most important viral pathogens in humans. Surface (S) protein is the major HBV antigen that mediates virus attachment and entry and determines the virus subtype. Mutations in S gene, particularly in the “a” determinant, can influence virus detection by ELISA and may generate escape mutants. Since no records have documented the S gene mutations in HBV strains circulating in Saudi Arabia, the current study was designed to study sequence variation of S gene in strains circulating in Saudi Arabia and its correlation with clinical and risk factors. Patients and Methods: A total of 123 HBV-infected patients were recruited for this study. Clinical and biochemical parameters, serological markers, and viral load were determined in all patients. The entire S gene sequence of samples with viral load exceeding 2000 IU/mL was retrieved and exploited in sequence and phylogenetic analysis. Patients and Methods: A total of 123 HBV-infected patients were recruited for this study. Clinical and biochemical parameters, serological markers, and viral load were determined in all patients. The entire S gene sequence of samples with viral load exceeding 2000 IU/mL was retrieved and exploited in sequence and phylogenetic analysis. Results: A total of 48 mutations (21 unique) were recorded in viral strains in Saudi Arabia, among which 24 (11 unique) changed their respective amino acids. Two amino acid changes were recorded in “a” determinant, including F130L and S135F with no evidence of the vaccine escape mutant G145R in any of the samples. No specific relationship was recognized between the mutation/amino acid change record of HBsAg in strains in Saudi Arabia and clinical or laboratory data. Phylogenetic analysis categorized HBV viral strains in Saudi Arabia as members of subgenotypes D1 and D3. Conclusion: The present report is the first that describes mutation analysis of HBsAg in strains in Saudi Arabia on both nucleotide and amino acid levels. Different substitutions, particularly in major hydrophilic region, may have a potential influence on disease diagnosis, vaccination strategy, and antiviral chemotherapy.

Development of potent vaccine for human respiratory syncytial virus (HRSV) that confers better protection than natural infection remains a global challenge. Vaccination with naked DNA is considered successful approach for the control of many viral diseases. In this study, the potential of DNA vaccination using full-length attachment gene of HRSV type A Saudi strain cloned in pcDNA3.1+ vector (pcDNA/GA) was evaluated in BALB/c mice. The expression efficiency of pcDNA/GA was first confirmed in HEp-2 cells on RNA and protein levels. Mice immunization with either pcDNA/GA or the positive control formalin-inactivated vaccine (FI-RSV) has generated significant serum antibody concentration in ELISA (7.31±0.418 and 9.76±0.006 µg/ml, respectively) with superior neutralizing activity. Similarly, both immunogens evoked robust HRSV-specific CD8+ T-cell response in ELISPOT assay compared to mice immunized with pcDNA3.1+ vector or saline (negative controls). Challenge of the immunized mice with the wild-type HRSV did not provoke clinical symptoms or mortality in any mice group. On the 7th day post-challenge, mice were euthanized and lungs were extirpated for evaluation of viral load, histopathological changes and cytokine profile. A significant diminish in the viral load and histology score were concluded in lungs of pcDNA/GA immunized mice compared to those immunized with FI-RSV and negative controls. The pulmonary cytokine profile of pcDNA/GA immunized mice displayed notable upregulation of Th1-associated cytokines while that of FI-RSV immunized mice exhibited high levels of Th2-associated cytokines. In conclusion, the DNA vaccine candidate pcDNA/GA has proven prominent efficacy and safety in mouse model, which encourages further evaluation in clinical trials.

Respiratory tract infections are a principal cause of illness and mortality in children worldwide and mostly caused by viruses. In this study, the epidemiology of 11 respiratory RNA viruses was investigated in a cohort of hospitalized children at a tertiary referral center in Riyadh from February 2008 to March 2009 using conventional and real-time monoplex RT-PCR assays. Among 174 nasopharyngeal aspirates, respiratory syncytial virus (RSV) was detected in 39 samples (22.41%), influenza A virus in 34 (19.54%), metapneumovirus (MPV) in 19 (10.92%), coronaviruses in 14 (8.05%), and parainfluenza viruses (PIVs) in 11 (6.32%). RSV, PIVs and coronaviruses were most prevalent in infants less than 6 months old, whereas MPV and influenza A virus were more prominent in children aged 7-24 and 25-60 months, respectively. The majority of the viruses were identified during winter with two peaks observed in March 2008 and January 2009. The presented data warrants further investigation to understand the epidemiology of respiratory viruses in Saudi Arabia on spatial and temporal basis.

Many types of naturally growing plants are used traditionally for the treatment of different types of cancers and infectious diseases. In this report, the cytotoxic activity of 30 medicinal plants, commonly used in folk medicine in Saudi Arabia and Indonesia, was evaluated in vitro using Vero and HEp-2 cell lines. Plants were randomly chosen and harvested from different districts of both countries based on ethnobotanical information and subsequently extracted by methanol. Serial two-fold dilutions of each extract, starting from the concentration of 1000 µg/ml, were incubated with Vero and HEp-2 cells for 72 h. The cytotoxic effect of different extracts was in vitro characterized by identification of cellular alterations microscopically and cellular viability colormetrically. The plant extracts were classified according to the minimal toxic concentration and 50% cytotoxicity concentration indexes into three groups: highly cytotoxic (8 to 31 µg/ml), moderately cyototoxic (32 to 499 µg/ml) and low cyototoxic (500 to 1000 µg/ml). The results showed that the extracts of Juniperus phoenicea and Calotropis procera were highly cytotoxic on both cell lines to the minimal concentration of 1 µg/ml and may be well-considered as potential candidates for anticancer research. Two more extracts (Datura inoxia and Citrullus colocynthis) produced significant cytotoxicity to the minimum concentration of 16 µg/ml, with selective powerful activity of C. colocynthis on HEp-2 cells. The other extracts showed lower degrees of cytotoxicity and may be utilized for testing as antiviral agents using cell culture models. Key words: Calotropis procera, cell titer blue assay, cytotoxic activity, Juniperus phoenicea, medicinal plants, methanolic extracts.

The genetic variability and circulation pattern of human respiratory syncytial virus group B (HRSV-B) strains, identified in Riyadh during the winters of 2008 and 2009, were evaluated by partial sequencing of the attachment (G) protein gene. The second hypervariable region (HVR-2) of G gene was amplified by RT-PCR, sequenced and compared to representatives of different HRSV-B genotypes. Sequence and phylogenetic analysis revealed that all Saudi strains belonged to the genotype BA, which is characterized by 60-nucleotide duplication at HVR-2. Only strains of 2008 were clustered with subgroup BA-IV, while those isolated at 2009 were clustered among the most recent subgroups (particularly BA-X and CB-B). Amino acid sequence analysis demonstrated 18 amino acid substitutions in Saudi HRSV-B strains; among which five are specific for individual strains. Furthermore, two potential N-glycosylation sites at residues 230 and 296 were identified for all Saudi strains, and an additional site at amino acid 273 was found only in Riyadh 28/2008 strain. O-glycosylation was predicted in 42-43 sites, where the majority (no = 38) are highly conserved among Saudi strains. The average ratio between non-synonymous and synonymous mutations (ω) implied stabilizing selection pressure on G protein, with evidences of positive selection on certain Saudi strains. This report provides preliminary data on the circulation pattern and molecular characteristics of HRSV-B strains circulating in Saudi Arabia.

Human metapneumovirus (HMPV) is an important cause of respiratory tract illness in children. Two HMPV subgroups, A and B, and four genotypes, A1, A2, B1 and B2, have been identified. Concurrent circulation of the different genotypes in yearly epidemics has been recorded globally, but not in Saudi Arabia. The current report was designed to study HMPV epidemiology in Saudi children and to analyze the genetic diversity and circulation patterns. Nasopharyngeal aspirates (n = 174) were collected from hospitalized children in Riyadh (2008-2009). The screening of samples using real-time RT-PCR identified 19 HMPV strains. The majority of the strains belonged to subgroup B, while all strains of subgroup A were members of genotype A2. In 2008, only subgroup B was recognized, whereas in 2009 both subgroups were identified to be cocirculating at similar rates. The full-length attachment (G) gene and a partial sequence of the fusion (F) gene of positive samples were sequenced. The G gene showed a high degree of genetic diversity and exhibited a variable number of positively selected sites in different lineages. In contrast, the F gene demonstrated an extensive genetic stability with a higher tendency toward purifying selection. This is the first report on HMPV genotype circulation in Saudi Arabia; however, the exact circulation kinetics requires further retrospective and prospective study.

A novel two-step, SYBR Green I based real-time RT-PCR assay was developed for detection and quantification of BCoV using ABI PRISM 7500 sequence detection system. The assay was carried out using two sets of primers designed to amplify highly conserved sequences of the nucleocapsid gene of BCoV and the internal control, bovine glyceraldehyde-3-phosphate dehydrogenase, RNA. Specific identification of both targets was elucidated by melt curve analysis, in which the BCoV amplified product generated a melt peak at 78.35 ± 0.26 °C and the internal control RNA at 82.54 ± 0.32 °C. The assay was highly specific since all negative controls and other viruses of clinical and structural relevance failed to develop any positive results. The detection limit of the reaction was 10(3) plasmid copies and 1.17 × 10(-3) TCID(50) of the tissue culture propagated virus. Standard deviation and coefficient of variation was low for both intra-assay and inter-assay variability. The assay performance on field samples was evaluated on 103 (68 fecal and 35 nasal) swab specimens and compared with the conventional RT-PCR assay. The results of both assays matched for the diagnosis of 65 fecal and 33 nasal samples. However, three fecal and two nasal samples tested negative in gel-based assay were positive for the real-time RT-PCR. The robustness and a high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and in BCoV research.

The use of embryonating chicken eggs in preparation of avian virus vaccines is the principle cause for contamination with Chicken anemia virus (CAV). Identification of CAV in contaminated vaccines relies on the expensive, tedious, and time-consuming practice of virus isolation in lymphoblastoid cell lines. The experience of the last 2 decades indicates that polymerase chain reaction is extending to replace most of the classic methods for detection of infectious agents. In the present report, a simple, rapid, and accurate polymerase chain reaction method for detection of CAV in poultry vaccines is described. Oligonucleotide primers homologous to highly conserved sequences of the VP1 gene were used to amplify a fragment of 676 bp. The developed assay was specific for detecting CAV from different sources, with no cross reactivity with many avian viruses. No inter- and intra-assay variations were observed. The analytical sensitivity of the test was high enough to detect 5 TCID50 (50% tissue culture infective dose) of the virus per reaction; however, different factors related to the vaccine matrix showed considerable effects on the detection limit. In conclusion, this method may represent a suitable alternative to virus isolation for identification of CAV contamination of poultry virus vaccines.

In the current study, two RT-PCR systems for detection (mono-specific) and typing (duplex) of RSV in clinical samples have been developed and validated. The two assays were exploited for studying the dissemination of RSV infection in Riyadh, Saudi Arabia during season 2007/2008. Furthermore, the effect of certain risk factors, including gender, age and disease form, on extended and impact of the infection was analyzed. Of two hundreds collected samples, 70(35%) were positive for RSV infection, among which 40 (%&.1%) were type A and 30 (42.9 %) were type B viruses. This investigation is the first that describes the existence of both RSV subtypes in co-ordinance in Saudi Arabia children, with slight dominance of type A viruses. By analyzing the effect of risk factors on the disease epidemiology we confirmed that males, patients at young age and asthmatics are more susceptible for RSV infection and disease progression. keywords: Respiratiry synctial virus, RT-PCR, typing, risk factors, Saudi Arabia abbreviation: CEBRPubs URL: http://coebio.ksu.edu.sa/publication/?doShow=abstract&pubID=9 For futher details, please contact us at coebio@ksu.edu.sa or +966 1 46 76418 .

Studies have indicated variations in the degree of efficacy of the commercial disinfectants commonly used in poultry production facilities. An adequate method of in vitro testing was used to compare the efficacy of some of these disinfectants while testing them in conditions similar to those of the poultry facilities. Five commercially available disinfectants were tested against 7 selected bacterial, fungal and viral isolates. The obtained results indicated that, most of the tested disinfectant products were effective at the manufacturer recommended level within 30 min contact time when tested in the absence of organic matter. However, when organic matter was present longer contact times were needed to demonstrate the effectiveness. Pseudomonas aeruginosa, Fusarium species and Newcastle disease virus showed variable degrees of resistance to some of the tested disinfectant products in the presence of organic matter. Conclusively, monitoring program should be adopted regularly in poultry facilities to test the problematic microbes individually for their resistance against commercial disinfectants.

Respiratory tract infections are a principal cause of illness and mortality in children worldwide and mostly caused by viruses. In this study, the epidemiology of 11 respiratory RNA viruses was investigated in a cohort of hospitalized children at a tertiary referral center in Riyadh from February 2008 to March 2009 using conventional and real‐time monoplex RT‐PCR assays. Among 174 nasopharyngeal aspirates, respiratory syncytial virus (RSV) was detected in 39 samples (22.41%), influenza A virus in 34 (19.54%), metapneumovirus (MPV) in 19 (10.92%), coronaviruses in 14 (8.05%), and parainfluenza viruses (PIVs) in 11 (6.32%). RSV, PIVs and coronaviruses were most prevalent in infants less than 6 months old, whereas MPV and influenza A virus were more prominent in children aged 7–24 and 25–60 months, respectively. The majority of the viruses were identified during winter with two peaks observed in March 2008 and January 2009. The presented data warrants further investigation to understand the epidemiology of respiratory viruses in Saudi Arabia on spatial and temporal basis. J. Med. Virol. 88:1086–1091, 2016. © 2015 Wiley Periodicals, Inc.

Influenza viruses are known as continuing threats to human public health every year worldwide. Evolutionary dynamics of influenza B viruses in humans are in a unique progression having two lineages; B/Yam and B/Vic-like viruses, which are circulating simultaneously worldwide. There is a considerable lack of data on influenza B viruses circulating in Saudi Arabia. During the winter-spring season of 2010-2011, 80 nasopharyngeal aspirates were collected from hospitalized patients with flu-like symptoms in Riyadh. Screening of samples by one-step RT-PCR identified three (3.8%) influenza B viruses. Sequencing of hemagglutinin (HA) and neuraminidase (NA) genes was performed to analyze influenza B viruses circulating in Riyadh as compared to the globally circulating strains. Several common and six unique amino acid substitutions were observed for both HA and NA genes of influenza B Saudi strains. Three unique substitutions (T182A, D196N, and K254R) were identified in HA gene of the B/Yam-like Riyadh strains. In NA gene, a unique common substitution (D53G) was found in all Riyadh strains, while two unique substitutions (L38P, G233R) were recognized only in B/Vic-like Riyadh strains. Riyadh strains were also found to contain N-glycosylation site in HA gene of both B/Vic and B/Yam lineages at positions 197-199 (NET) and 196-198 (NNK/DNK), respectively. The significance of these mutations on the antigenicity of both lineages is discussed herein. The unique changes observed in HA and NA genes of influenza B Riyadh strains support strongly the need for continuous surveillance and monitoring of new evolving strains that might pose threat to the Saudi community.

Human respiratory syncytial virus (HRSV) is a frequent cause of hospitalization and mortality in children worldwide. The molecular epidemiology and circulation pattern of HRSV in Saudi Arabia is mostly uncharted. In the current study, the genetic variability and phylogenetic relationships of HRSV type A strains circulating in Riyadh Province were explored. Nasopharyngeal aspirates were collected from hospitalized children with acute respiratory symptoms during the winter-spring seasons of 2007/08 and 2008/09. Among 175 samples analyzed, 39 (22.3 %) were positive for HRSV by one-step RT-PCR (59 % type A and 41 % type B). Propagation of positive samples in HEp-2 cells permitted the recovery of the first Saudi HRSV isolates. Genetic variability among Saudi HRSV-A strains was evaluated by sequence analysis of the complete attachment (G) protein gene. The nucleotide sequence was compared to representatives of the previously identified HRSV-A genotypes. Sequence and phylogenetic analysis showed that the strains examined in this study were very closely related at both the nucleotide and amino acid level, and all of them are clustered in the GA2 genotype (and mostly belonged to the NA-1 subtype). A total of 23 mutation sites, 14 of which resulted in an amino acid change, were recorded only in Saudi strains. This is the first report on genetic diversity of HRSV-A strains in Saudi Arabia. Further analysis of strains on a geographical and temporal basis is needed to fully understand HRSV-A circulation patterns in Saudi Arabia.

Highlights • A real time recombinase polymerase amplification assay was developed for the detection of bovine coronavirus. • The assay was rapid (10–20 min), with analytical sensitivity of 19 RNA molecules and specific. • The combination of this assay with a new portable fluorescence reader indeed is a new step toward point-of-care nucleic acid detection., Bovine coronavirus (BCoV) is an economically significant cause of calf scours and winter dysentery of adult cattle, and may induce respiratory tract infections in cattle of all ages. Early diagnosis of BCoV helps to diminish its burden on the dairy and beef industry. Real-time RT-PCR assay for the detection of BCoV has been described, but it is relatively expensive, requires well-equipped laboratories and is not suitable for on-site screening. A novel assay, using reverse transcription recombinase polymerase amplification (RT-RPA), for the detection of BCoV is developed. The BCoV RT-RPA was rapid (10–20 min) and has an analytical sensitivity of 19 molecules. No cross-reactivity with other viruses causing bovine gastrointestinal and/or respiratory infections was observed. The assay performance on clinical samples was validated by testing 16 fecal and 14 nasal swab specimens and compared to real-time RT-PCR. Both assays provided comparable results. The RT-RPA assay was significantly more rapid than the real-time RT-PCR assay. The BCoV RT-RPA constitutes a suitable accurate, sensitive and rapid alternative to the common measures used for BCoV diagnosis. In addition, the use of a portable fluorescence reading device extends its application potential to use in the field and point-of-care diagnosis.

Background Although human parainfluenza type 2 (HPIV-2) virus is an important respiratory pathogen, a little is known about strains circulating in Saudi Arabia. Findings Among 180 nasopharyngeal aspirates collected from suspected cases in Riyadh, only one sample (0.56%) was confirmed HPIV-2 positive by nested RT-PCR. The sample that was designated Riyadh 105/2009 was used for sequencing and phylogenetic analysis of the most variable virus gene; the haemagglutinin-neuramindase (HN). Comparison of HN gene of Riyadh 105/2009 strain and the relevant sequences available in GenBank revealed a strong relationship with Oklahoma-94-2009 strain. Phylogenetic analysis indicated four different clusters of HPIV-2 strains (G1-4). Twenty-three amino acid substitutions were recorded for Riyadh 105/2009, from which four are unique. The majority of substitutions (n=18) had changed their amino acids characteristics. By analyzing the effect of the recorded substitutions on the protein function using SIFT program, only two located at positions 360 and 571 were predicted to be deleterious. Conclusions The presented changes of Riyadh 105/2009 strain may possess potential effect on the protein structure and/or function level. This is the first report that describes partial characterization of Saudi HPIV-2 strain.

Human parainfluenza virus 3 (HPIV-3) is a leading cause of respiratory disease in children worldwide. Previous sequence analyses of the entire virus genome, among different HPIV-3 strains, demonstrated that HN is the most variable gene. There is a dearth of data on HPIV-3 strains circulating in Saudi Arabia. In this report, HPIV-3 was screened in nasopharyngeal aspirates collected from hospitalized children with acute respiratory disease during two successive seasons (2007/08 and 2008/09) using nested RT-PCR. Out of 73 samples collected during 2007/08, seven (9.59%) were positive; while 3 out of 107 samples collected during 2008/09 (2.8%) were positive. Virus isolation in cell culture was successful using HEp2, but not Vero cells. The identity of the isolated viruses was confirmed using immunofluorescence and neutralization assays. To elucidate the genetic characteristics and phylogeny of Saudi HPIV-3 strains, the complete HN gene sequence of two selected Saudi strains was analyzed in comparison to 20 strains isolated by others from different countries worldwide. Both strains showed the highest degree of sequence homology with Indian strains, followed by Chinese and most Japanese strains. Phylogenetic analysis confirmed that these strains fell into a distinct Asian lineage. This study is the first in Saudi Arabia to recover HPIV-3 isolates of confirmed identity, and to generate sequence data that may help in understanding virus diversity and evolution.

The Egyptian Arabian horse is one of the oldest and most popular horse breeds in the world. No previous efforts have been made for investigating the existence and prevalence of equine herpes viruses (EHVs) in this precious horse breed. In this report, ninety three clinical samples were collected from a cohort of Arabian horses located in Cairo, Egypt. Screening of the clinical samples for the presence of EHV antigens by cell-ELISA utilizing a polyclonal antibody pool against EHV-1, 2, and 4 identified 34 (36.56%) positive samples. Virus-specific semi-nested PCR assays were used for typing the positive samples. Three samples were found positive for EHV-1, seventeen for EHV-2, seven for EHV-4, one was a mixed infection of EHV-1 and EHV-4, and six did not produce any amplification signal with all assays. Sequence analysis of the amplified semi-nested PCR products of representative EHV strains further confirmed the virus identity. This study is the first that outlines the involvement of EHV infections in Arabian horse diseases worldwide. Besides, it presents new data about the prevalence of EHVs in the Egyptian horse population. Key words: Arabian horses, Egypt, equine herpes viruses, prevalence.

Interferon (IFN) is produced and secreted by various mammalian cell lines when they are infected by viruses, and it plays a regulatory role in innate immunity against viral infections. It also represents a significant therapeutic molecule in a number of viral diseases and cancers. IFN induces the Jak/STAT pathway leading to the activation and binding of transcriptional activators, e.g., the STAT/IRF9 complex, to the IFN-stimulated response element (ISRE) in the promoters of IFN-stimulated genes (2, 18). The transcription of IFN genes also is mediated via specific virus response elements (VREs) in the promoter; these sequences bind different IFN response factors (IRFs), such as IRF-3 and IRF-7, in the promoters of IFN genes (17). The VRE and ISRE sequences are found in IFN genes and IFN-stimulated genes; they partially overlap with each other, particularly the core AANNGAAA with the following consensus: G(A)AAANNGAAAG/CT/C or A/GNG AAANNGAAACT (also in the complementary strand) (8, 19). Hundreds of virus- and IFN-stimulated genes exist in the human genome (15), and although their promoters harbor specific core sequence consensus elements, these sequences have context heterogeneity, variable reiterations, and distinct transactivation potential. These sequence variations may account for responses to various types of viruses, IFNs, and IRFs. In this study, we have developed and validated a live cell-based enhanced green fluorescent protein (EGFP) reporter system employing 120 constructs containing multiple viruses and IFN response reporters that represent IFN system nucleic acid sequence heterogeneity to monitor and assess promoter sequence-function relationships during innate immunity. Several sequence reporters with highly sensitive and early activation were observed with distinct patterns of responses to different ligands, including IFN, double-stranded RNA, and RNA viruses.

ZH501 strain of Rift valley fever virus (RVFV) was originally isolated from a human patient during the outbreak of 1977 in Egypt. This virus strain was used since 1980 for preparation of an inactivated RVF vaccine at Veterinary Serum and Vaccine Research Institute (VSVRI), Egypt. Two subpopulations of ZH501 with a single nucleotide polymorphism (A/G) at nucleotide position 847 of Gn gene of M segment have been described. This nucleotide substitution affected significantly RVFV virulence in the mouse model. In this report, the genetic makeup of the Gn gene of ZH501-VRVSI vaccine virus stock was analyzed for verification of its safety and stability. Plaque assay of the vaccine stock virus revealed the presence of two populations that produced different plaque forms. The viruses that developed large plaques resembling those produced by the virulent virus strains were isolated for genetic analysis. Comparison of the Gn gene nucleotide sequence of the isolated viruses with those of the wild-type ZH501 and other reference strains identified five nucleotide substitutions, of which three are capable to induce amino acid changes in the mature protein. Protean analysis suggested a potential change in the three dimensional structure of the Gn protein in relation to the parent strain. The results of this study throw light on the changes occurred in the master seed virus used for preparation of RVFV vaccine in Egypt. Further studies focusing on the other gene segments of ZH501-VSVRI are required to conclude is it still the best choice for vaccination against RVF in Egypt.

Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism.