Haitham Amer

BACKGROUND: Equine influenza (EI) is a transmissible viral respiratory sickness of the 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.

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.

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 10 50% tissue culture infective dose (TCID) 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 log). 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.

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.

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.

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 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: 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.

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 ( = 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 ( = 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.

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 NA-1 to ON-1, which was prevalent during the winter seasons of 2007/08 and 2008/09. Based on the inter-genotypic 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 NA-1 and NA-2 subgenotypes in Saudi Arabia with ON-1 within 7-8 years. The continuous evolution of Human orthopneumovirus through point mutations and nucleotide duplication may explain its ability to cause recurrent infections. This article is protected by copyright. All rights reserved.

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.

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.

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. Te expression efciency of pcDNA/GA was frst confrmed 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 signifcant 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-specifc 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 profle. A signifcant 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. Te pulmonary cytokine profle of pcDNA/GA immunized mice displayed
notable upregulation of T1-associated cytokines while that of FI-RSV immunized mice exhibited high levels
of T2-associated cytokines. In conclusion, the DNA vaccine candidate pcDNA/GA has proven prominent
efcacy and safety in mouse model, which encourages further evaluation in clinical trials.

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 codonoptimized 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 CD8C 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

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.

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.

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.

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.

UNLABELLED: Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with >240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN-γ activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE- and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN response-betacellulin (BTC), interleukin 11 (IL-11), and IL-17F-that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-β and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-β response through the NF-κB pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-α against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus, in susceptible cell lines. Overall, the study represents a large-scale analysis of cytokines for enhancing the IFN response and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities.

IMPORTANCE: Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN), which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

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.

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.

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 (x) 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.

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.