Assessment of the potency and effectiveness of a heptavalent oil-adjuvanted (ISA 206) foot-and-mouth disease vaccine in Egypt.

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.

Emergence of equine influenza virus H3Nx Florida clade 2 in Arabian racehorses in Egypt.

BACKGROUND: Equine influenza is an important cause of respiratory disease in equids. The causative virus; EIV, is highly variable and can evolve by accumulation of mutations, particularly in the haemagglutinin (HA) gene. Currently, H3N8 is the sole subtype circulating worldwide with Florida clade 1 (FC1) is most prevalent in the Americas and FC2 in Asia and Europe. In Egypt, EIV was detected in two occasions: subtype H7N7 in 1989 and subtype H3N8 (FC1) in 2008. No data is available on the circulation pattern of EIV during the last decade despite frequent observation of suspected cases. METHODS: Twenty-two nasal swabs were collected from vaccinated and non-vaccinated horses showing respiratory signs suggestive of EIV infection in 2017-18. Three additional swabs were retrieved during a national race event in January 2018 from Arabian mares with high fever, gait stiffness and dry cough. Samples were screened by RT-qPCR and HA1 domain of the hemagglutinin gene was amplified and sequenced for sequence and phylogenetic analysis. RESULTS: RT-qPCR screening revealed that only the 3 samples from the race were positive with cycle thresholds ranging from 16 to 21 indicating high viral load. Isolation attempts in hen's eggs were unsuccessful. Sequence analysis of the HA1 domain gene has revealed two identical nucleotide sequences, while the third contained 3 synonymous mutations. Phylogenetic analysis clustered study sequences with recent FC2 sequences from Europe. Amino acid alignments revealed 14 and 13 amino acid differences in the study sequences compared to A/equine/Egypt/6066NANRU-VSVRI/08 (H3N8) and A/equine/Kentucky/1997 (H3N8), respectively, available as EIV vaccines in Egypt. Nine amino acids were different from A/equine/Richmond/1/2007 (H3N8), the recommended FC2 vaccine strain by the world organization of animal health expert surveillance panel (OIE-ESP), two of which were unique to the Egyptian sequences while the remaining 7 changes were shared with the FC2-144V subgroup detected in the United Kingdom from late 2015 to 2016. CONCLUSIONS: The study represents the first reported detection of FC2-144V related EIV from Arabian mares in Egypt, and probably from the entire middle east region. The presented information about EIV epidemiology and spread may require reconsideration of the vaccine strains used in the national vaccination programs.

Latest Insights on the Diagnostic Approaches and Treatment Strategies of COVID-19.

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.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

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.

Sequence and phylogentic analysis of MERS-CoV in Saudi Arabia, 2012-2019.

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.

Gallid Alphaherpesvirus 2 in the Egyptian Turkeys: Molecular Characterization and Establishment of a Universal System for Phylogenetic Classification.

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.

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.

Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay.

Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Molecular diagnostic techniques like RT-PCR and real-time RT-PCR are considered the gold standard for identification of H5 influenza viruses in clinical samples. These techniques are hampered by the need of well-equipped laboratories, large space requirement, and relatively long time-to-result. Recombinase polymerase amplification (RPA) assay represents an excellent alternative to PCR since it is more simple, rapid, economic, and portable. Reverse transcription RPA (RT-RPA) assay was recently developed for sensitive and specific detection of H5N1 virus in 6-10 min. To ensure the accuracy of the developed assay, two approaches for using a positive control were evaluated in this study. These approaches included: 1) all-in-one (internal positive control; IPC), 2) two-tubes-per-one-sample (external positive control; EPC). Sigma virus (SIGV) RNA and turkey mitochondrial DNA were tested as positive controls in both approaches. For all-in-one approach, both targets (H5 and IPC) were strongly inhibited. In contrast, very good amplification signals were obtained for the two types of EPC with no effect on the analytical sensitivity and specificity of H5 RT-RPA assay in two-tubes-per-one-sample approach. The performance of EPC-based H5 RT-RPA was further validated using 13 tracheal swabs. The results were compared to real-time RT-PCR and proved superior specificity in detecting H5N1 but not H5N8 viruses. Inclusion of EPC did not affect the aptitude of both assays in terms of sensitivity, specificity and reproducibility. In conclusion, the two-tubes-per-one-sample approach was more reliable to control the false negative results in H5 RT-RPA assay.

Molecular characterization and genetic diversity of the infectious laryngotracheitis virus strains circulating in Egypt during the outbreaks of 2018 and 2019.

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.

Epidemiology of respiratory viruses in Saudi Arabia: toward a complete picture.

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.

Epidemiology of 11 respiratory RNA viruses in a cohort of hospitalized children in Riyadh, Saudi Arabia.

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.

Hemagglutinin and neuraminidase genes of influenza B viruses circulating in Riyadh, Saudi Arabia during 2010-2011: evolution and sequence analysis.

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.

Genetic diversity in the G protein gene of group A human respiratory syncytial viruses circulating in Riyadh, Saudi Arabia.

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.

Group B strains of human respiratory syncytial virus in Saudi Arabia: molecular and phylogenetic analysis.

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.

Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Background: Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains. Aim: The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2. Methods: In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system. Results: The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays. Conclusion: In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

Molecular characterization of full-length VP2 gene of canine parvovirus type 2 strains circulating in Egypt 2019-2021.

Canine parvovirus type 2 (CPV-2) is a major cause of fatal gastroenteritis and myocarditis in puppies of domestic and wild carnivores. CPV-2 has accumulated changes over time lead to the emergence of three antigenic variants CPV-2a, CPV-2b, and CPV-2c. VP2 is the major capsid protein that determines virus antigenicity, and host range. Although the three CPV-2 variants were previously identified in Egypt, most reports covered a restricted geographic region and/or time period, and only analyzed partial fragments of VP2 gene. Therefore, this study was designed to test 100 rectal swabs collected from 7 Egyptian governorates between 2019 and 2021 for CPV-2 using PCR. A total of 65 positive samples were identified, mostly in pure dog breeds of young age. The three variants co-circulated in 2019, while CPV-2b was not detected in 2020 and 2021. The frequency of CPV-2b and CPV-2c was higher in 2019 and 2021, respectively. Analysis of CPV-2 full-length VP2 gene sequence from 19/65 positive samples has identified four common amino acid substitutions F267Y, S297A, A300G, Y324I, which are characteristic for the new CPV-2 variants currently circulating worldwide. Unique substitutions including A5G, G36R, V38E, Q370R, and G392V were recognized in certain samples, and appears to have distinct effect on receptor binding, nuclear translocation, and inter-species transmission. Phylogenetic analysis showed separation of CPV-2 strains into two clades. All strains of this study were classified in clade I with Asian strains. In conclusion, this study provides updated comprehensive molecular analysis of CPV-2 variants in Egypt.

Green fluorescent protein reporter system with transcriptional sequence heterogeneity for monitoring the interferon response.

The interferon (IFN) response is initiated by a variety of triggers, including viruses and foreign RNA, and involves several receptors and intracellular mediators. Although there are common cis-acting consensus sequences in the promoters of many genes stimulated during the IFN response, they exhibit core and context heterogeneity that may lead to differential transcriptional activity. We have developed and validated a live cell-based enhanced green fluorescent protein (EGFP) reporter system employing more than a hundred constructs containing multiple viruses and IFN response elements derived from a variety of promoters involved in immunity to viruses. Common and distinct response patterns were observed due to promoter heterogeneity in response to different stimuli, including IFN-α, TLR3-agonist double-stranded RNA, and several viruses. This information should serve as a resource in selecting specific reporters for sensing nonself ligands.

Molecular characterization and phylogenetic analysis of human parainfluenza virus type 3 isolated from Saudi Arabia.

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.

Human parainfluenza virus type 2 hemagglutinin-neuramindase gene: sequence and phylogenetic analysis of the Saudi strain Riyadh 105/2009.

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.