RESUMO
The extensive mass gathering of pilgrims from all over the world, as well as the constant flow of foreign workers via country entry crossings, raises the likelihood of respiratory virus outbreaks spreading and evolving in Saudi Arabia. Here, we report the sequence and phylogenetic analysis of the human parainfluenza type-2 (HPIV-2) in nasopharyngeal aspirates (NPAs) collected from Riyadh, Saudi Arabia, from 2020/21 to 2021/22 seasons. RNA was extracted from the clinical samples and subjected to RT-PCR analysis for the detection of IAV and IBV. The full-length HN gene of HPIV-2 was amplified and sequenced. Multiple sequence alignments (both nucleotides and deduced amino acids) were aligned using Clustal W, MegAlign program of Lasergene software, and MEGA 7.0. HPIV-2 was found in (4; 2% of 200) NPAs. Sequence and phylogenetic analysis results showed that indicated a genotype shifting from G3 to G4a with 83% sequence homology 62-M786 from Japan, which was prominent throughout the winter seasons of 2008/09. Multiple amino acid sequence alignment revealed 25 sites of possible difference between G3 genotypes and G4a. A total of twenty- two of these locations were shared by the other G4a genotypes, whereas three positions, 67 V, 175 S, and 377Q, were exclusively shared by G3. Only eight conserved N-glycosylation sites were found at amino acids 6(NLS), 286(NTT), 335(NIT), 388(NNS), 498(NES), 504(NPT), 517(NTT), and 539(NGT) in four Riyadh isolates. Our findings also revealed that the G4a genotype of HPIV-2 predominated in our samples population during the winter seasons of 2020/21 and 2021/22. Further research with a larger sample size covering numerous regions of Saudi Arabia throughout different epidemic seasons is needed to achieve an improved knowledge of HPIV-2 circulation.
Assuntos
Infecções por Paramyxoviridae , Humanos , Arábia Saudita/epidemiologia , Filogenia , Sequência de Aminoácidos , Aminoácidos/genética , Vírus da Parainfluenza 1 Humana , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 2 HumanaRESUMO
Evolutionary studies on Dengue virus (DENV) in endemic regions are necessary since naturally occurring mutations may lead to genotypic variations or shifts in serotypes, which may lead to future outbreaks. Our study comprehends the evolutionary dynamics of DENV, using phylogenetic, molecular clock, skyline plots, network, selection pressure, and entropy analyses based on partial CprM gene sequences. We have collected 250 samples, 161 in 2017 and 89 in 2018. Details for the 2017 samples were published in our previous article and that of 2018 are presented in this study. Further evolutionary analysis was carried out using 800 sequences, which incorporate the study and global sequences from GenBank: DENV-1 (n = 240), DENV-3 (n = 374), and DENV-4 (n = 186), identified during 1944-2020, 1956-2020, and 1956-2021, respectively. Genotypes V, III, and I were identified as the predominant genotypes of the DENV-1, DENV-3, and DENV-4 serotypes, respectively. The rate of nucleotide substitution was found highest in DENV-3 (7.90 × 10-4 s/s/y), followed by DENV-4 (6.23 × 10-4 s/s/y) and DENV-1 (5.99 × 10-4 s/s/y). The Bayesian skyline plots of the Indian strains revealed dissimilar patterns amongst the population size of the three serotypes. Network analyses showed the presence of different clusters within the prevalent genotypes. The data presented in this study will assist in supplementing the measures for vaccine development against DENV.
Assuntos
Vírus da Dengue , Dengue , Humanos , Vírus da Dengue/genética , Sorogrupo , Dengue/epidemiologia , Filogenia , Teorema de Bayes , GenótipoRESUMO
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children and infants. To date, there is no effective vaccine available against RSV. Heparan sulfate is a type of glycosaminoglycan that aids in the attachment of the RSV to the host cell membrane via the G protein. In the present study, the effect of amino acid substitution on the structure and stability of the ectodomain G protein was studied. Further, it was investigated whether mutation (K117A) in the CX3C motif of G protein alters the binding with heparan sulfate. The point mutation significantly affects the conformational stability of the G protein. The mutant protein showed a low binding affinity with heparan sulfate as compared to the wild-type G protein, as determined by fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking studies. The low binding affinity and decreased stability suggested that this mutation may play an important role in prevention of attachment of virion to the host cell receptors. Collectively, this investigation suggests that mutation in the CX3C motif of G protein may likely improve the efficacy and safety of the RSV vaccine.
Assuntos
Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Heparitina Sulfato/metabolismo , Mutação/genética , Substituição de Aminoácidos/genética , Calorimetria/métodos , Linhagem Celular , Humanos , Simulação de Acoplamento Molecular/métodos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Análise Espectral/métodos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/genética , Replicação Viral/genéticaRESUMO
Respiratory tract infections are the primary cause of morbidity and mortality globally. Human bocavirus 1 (HBoV1), a member of the Parvoviridae family causes a wide spectrum of respiratory diseases in children, and gastroenteritis in adults. The mechanisms of latency, persistence, and reinfection of Bocavirus are poorly understood at present due to the lack of permissive cell lines and efficient animal models. Moreover, the dual infections of HBoV and other respiratory viruses further complicate the study of the pathogenicity of Bocaviruses. The data on immunological consequences of Bocavirus infection are sparse. However, the existing data have highlighted the role of CD4 T cells in Bocavirus infection. High titres of HBoV-specific antibodies have been detected in different populations suggesting its ubiquitous prevalence. Interestingly, the mechanism employed by Bocavirus to evade the immune system mostly targets type I IFN pathways and cause pyroptotic cell death of host cells. This review summarizes the immune responses evoked in response to Bocavirus infection, escape mechanism employed by the virus, and the vaccination strategies, including antisense technology to combat Bocavirus infections.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Bocavirus Humano/imunologia , Evasão da Resposta Imune , Imunidade , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , Animais , Humanos , Imunidade Celular , Imunidade Humoral , Infecções por Parvoviridae/prevenção & controle , Vacinas Virais/imunologiaRESUMO
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.
Assuntos
Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Códon/genética , Biologia Computacional , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/prevenção & controle , Evolução Molecular , Genômica , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Mutação , Fases de Leitura Aberta/genética , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Arábia Saudita , Escarro/virologiaRESUMO
BACKGROUND: Methotrexate (MTX) is an antirheumatic drug, transported by reduced folate carrier-1 (RFC1). The most common RFC1 gene variant, c.80 A>G (rs1051266) is ambiguously linked to adverse effects of MTX therapy in some rheumatoid arthritis (RA) patients. OBJECTIVE: The purpose of meta-analysis was to summarize all major published studies on c.80 A>G SNP to clarify this ambiguity in MTX therapy. METHODS: A total of 18 studies representing 3592 RA patients comprising 699 men and 2893 women were included. Both fixed and random effect models were applied to study the data. RESULTS: The RFC1 80A-allele showed null association with MTX-mediated toxicity in both fixed (odds ratio [OR] = 0.91; 95% CI = 0.80-1.03) and random effects (OR = 0.89; 95% CI: 0.71-1.11) models. Because heterogeneity was observed in this association (P = 0.0006), data were segregated based on use of folate therapy. In 7 studies (n = 1191) where folate was used along with MTX, RFC1 AA patients showed reduced risk for MTX-mediated toxicity (OR = 0.67; 95% CI: 0.50-0.89; P = 0.0006). The RFC1 80A-allele was found to increase the efficacy of MTX therapy by 1.53-fold (95% CI: 1.24-1.88), whereas the 80AA-genotype increased the efficacy by 1.85-fold (95% CI: 1.41-2.42). No publication bias was observed in these associations. CONCLUSION AND RELEVANCE: RFC1 c.80 A>G is an important pharmacogenetic determinant of MTX therapy in RA. The RFC1 80A-allele robustly increased therapeutic efficacy and safety when folate was used along with MTX. Findings are relevant to decision-making in the clinical use of MTX as a treatment for RA patients harboring the RFC1 gene variant.
Assuntos
Antirreumáticos , Artrite Reumatoide , Antirreumáticos/efeitos adversos , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Feminino , Genótipo , Humanos , Masculino , Metotrexato/efeitos adversos , Polimorfismo GenéticoRESUMO
The global burden of disease caused by a respiratory syncytial virus (RSV) is becoming more widely recognized in young children and adults. Heparan sulfate helps in attaching the virion through G protein with the host cell membrane. In this study, we examined the structural changes of ectodomain G protein (edG) in a wide pH range. The absorbance results revealed that protein maintains its tertiary structure at physiological and highly acidic and alkaline pH. However, visible aggregation of protein was observed in mild acidic pH. The intrinsic fluorescence study shows no significant change in the λmax except at pH 12.0. The ANS fluorescence of edG at pH 2.0 and 3.0 forms an acid-induced molten globule-like state. The denaturation transition curve monitored by fluorescence spectroscopy revealed that urea and GdmCl induced denaturation native (N) â denatured (D) state follows a two-state process. The fluorescence quenching, molecular docking, and 50 ns simulation measurements suggested that heparan sulfate showed excellent binding affinity to edG. Our binding study provides a preliminary insight into the interaction of edG to the host cell membrane via heparan sulfate. This binding can be inhibited using experimental approaches at the molecular level leading to the prevention of effective host-pathogen interaction.
Assuntos
Domínio Catalítico , Heparitina Sulfato/metabolismo , Interações Hospedeiro-Patógeno , Simulação de Acoplamento Molecular/métodos , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Membrana Celular/metabolismo , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Espectrometria de Fluorescência/métodos , Ureia/farmacologiaRESUMO
The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins.
Assuntos
Substâncias Macromoleculares/química , Mioglobina/química , Redobramento de Proteína , Temperatura , Animais , Difusão Dinâmica da Luz , Etilenoglicol/química , Fluorescência , Guanidina/farmacologia , Cavalos , Hidrodinâmica , Simulação de Acoplamento Molecular , Polietilenoglicóis/química , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Redobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacosRESUMO
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.
Assuntos
Filogenia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Mutação , Nasofaringe/virologia , Prevalência , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/virologia , Arábia Saudita/epidemiologia , Estações do Ano , Análise de Sequência de DNA , Fatores SexuaisRESUMO
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.
Assuntos
Infecções por Coronavirus/epidemiologia , Infecções Respiratórias/epidemiologia , Animais , Infecções por Coronavirus/virologia , Humanos , Islamismo , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Infecções Respiratórias/virologia , Arábia Saudita/epidemiologia , ViagemRESUMO
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-ß 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-ß 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.
Assuntos
Citocinas/biossíntese , Perfilação da Expressão Gênica , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Western Blotting , Linhagem Celular , Efeito Citopatogênico Viral , Vírus da Encefalomiocardite/imunologia , Humanos , Orthomyxoviridae/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Vesiculovirus/imunologiaRESUMO
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.
Assuntos
Criança Hospitalizada , Infecções por Vírus de RNA/epidemiologia , Infecções por Vírus de RNA/virologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Pré-Escolar , Coronavirus/genética , Coronavirus/isolamento & purificação , Estudos Transversais , Feminino , Humanos , Lactente , Recém-Nascido , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Masculino , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Nasofaringe/virologia , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 1 Humana/isolamento & purificação , Vírus Sincicial Respiratório Humano/genética , Arábia Saudita/epidemiologia , Estações do AnoRESUMO
The most comprehensive phylogenetic classification of human parainfluenza virus 3 (HPIV-3) was recently developed [PLoS One 2012;7:e43893]. This classification included three distinct clusters (A, B and C) with subdivision of cluster C into four subclusters (C1-4). In the present report, the classification of HPIV-3 was refined by inclusion of 27 overlooked beside newly characterized Saudi variants. The new phylogram was developed and included the same clusters described before, in which cluster A remained unchanged and cluster B contained more recent isolates. The organization of cluster C was altered through inclusion of a new subcluster (C5), subdivision of C1 into two lineages C1a and C1b and subdivision of C3 into three lineages C3a, C3b and C3c. The majority of Saudi variants were classified as members of subcluster C1b, whereas only one variant was placed in each of subclusters C2 and C5. This study illustrates an up-to-date phylogenetic classification of HPIV-3 variants.
Assuntos
Proteína HN/genética , Vírus da Parainfluenza 3 Humana/classificação , Vírus da Parainfluenza 3 Humana/genética , Sequência de Bases , Humanos , Filogenia , RNA Viral , Infecções por Respirovirus/virologiaRESUMO
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.
Assuntos
Variação Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza B/classificação , Vírus da Influenza B/genética , Influenza Humana/epidemiologia , Influenza Humana/virologia , Neuraminidase/genética , Proteínas Virais/genética , Substituição de Aminoácidos , Genótipo , Hospitalização , Humanos , Vírus da Influenza B/isolamento & purificação , Epidemiologia Molecular , Dados de Sequência Molecular , Nasofaringe/virologia , Filogenia , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Arábia Saudita/epidemiologia , Análise de Sequência de DNARESUMO
BACKGROUND: Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer. METHODS: In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 - the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems. RESULTS: High-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression. CONCLUSIONS: The fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs.
Assuntos
Vacinas Anticâncer/farmacologia , Proteínas E7 de Papillomavirus/imunologia , Vacinas contra Papillomavirus/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Adjuvantes Imunológicos/farmacologia , Animais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/genética , Vacinas contra Papillomavirus/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia , Zeína/imunologiaRESUMO
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.
Assuntos
Variação Genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Mutação , Filogenia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Arábia Saudita/epidemiologia , Estações do Ano , Alinhamento de SequênciaRESUMO
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.
Assuntos
Filogenia , Vírus Sinciciais Respiratórios/isolamento & purificação , Genes Virais , Glicosilação , Humanos , Vírus Sinciciais Respiratórios/genética , Arábia SauditaRESUMO
In the present work, a series of 0, 1 and 7 wt% silver nano-particles (Ag NPs) incorporated poly lactic-co-glycolic acid (PLGA) nano-fibers were synthesized by the electrospinning process. The PLGA/Ag nano-fibers sheets were characterized using SEM, TEM and DSC analyses. The three synthesized PLGA/silver nano-fiber composites were screened for anticancer activity against liver cancer cell line using MTT and LDH assays. The anticancer activity of PLGA nano-fibers showed a remarkable improvement due to increasing the concentration of the Ag NPs. In addition to the given result, PLGA nano-fibers did not show any cytotoxic effect. However, PLGA nano-fibers that contain 1 % nano silver showed anticancer activity of 8.8 %, through increasing the concentration of the nano silver to 7 % onto PLGA nano-fibers, the anticancer activity was enhanced to a 67.6 %. Furthermore, the antibacterial activities of these three nano-fibers, against the five bacteria strains namely; E.coli o157:H7 ATCC 51659, Staphylococcus aureus ATCC 13565, Bacillus cereus EMCC 1080, Listeria monocytogenes EMCC 1875 and Salmonella typhimurium ATCC25566 using the disc diffusion method, were evaluated. Sample with an enhanced inhibitory effect was PLGA/Ag NPs (7 %) which inhibited all strains (inhibition zone diameter 10 mm); PLGA/Ag NPs (1 %) sample inhibited only one strain (B. cereus) with zone diameter 8 mm. The PLGA nano-fiber sample has not shown any antimicrobial activity. Based on the anticancer as well as the antimicrobial results in this study, it can be postulated that: PLGA nanofibers containing 7 % nano silver are suitable as anticancer- and antibiotic-drug delivery systems, as they will increase the anticancer as well as the antibiotic drug potency without cytotoxicity effect on the normal cells. These findings also suggest that Ag NPs, of the size (5-10 nm) evaluated in the present study, are appropriate for therapeutic application from a safety standpoint.
Assuntos
Anti-Infecciosos/administração & dosagem , Antineoplásicos/administração & dosagem , Materiais Biocompatíveis/química , Sistemas de Liberação de Medicamentos , Prata/administração & dosagem , Linhagem Celular , Técnicas Eletroquímicas , Células Hep G2 , Humanos , Ácido Láctico/química , Neoplasias Hepáticas/tratamento farmacológico , Teste de Materiais , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Nanocompostos/química , Nanocompostos/ultraestrutura , Nanofibras/química , Nanofibras/ultraestrutura , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
Respiratory tract infections due to a variety of viruses continue to threaten the human population worldwide, particularly in developing countries. Among the responsible viruses, Human Bocavirus (HBoV), a novel discovered virus, causes respiratory tract and gastroenteritis disorders in young children. In Saudi Arabia, data regarding virus molecular epidemiology and evolution and its implication in respiratory tract infection are scarce. In the current study, genetic diversity and circulation pattern of HBoV-1 among hospitalized children due to acute respiratory tract infection (ARTI) during two consecutive years were charted. We found that 3.44% (2014/2015) and 11.25% (2015/2016) of children hospitalized due to ARTI were infected by HBoV-1. We have shown that HBoV was detected year-round without a marked seasonal peak. HBoV-1 also was co-detected with one or multiple other respiratory viruses. The multisequence analysis showed high sequence identity (â¼99%) (few point mutation sites) between strains of each genotype and high sequence variation (â¼79%) between HBoV-1 and the other 3 genotypes. Phylogenetic analysis showed the clustering of the study's isolates in the HBoV-1 subclade. Our data reveal that genetically conserved HBoV-1 was circulating among admitted children during the course of the study. Further epidemiological and molecular characterization of multiple HBoV-1 strains for different years and from all regions of Saudi Arabia are required to understand and monitor the virus evolution.
RESUMO
BACKGROUND: The replacement of hard tissues demands biocompatible and sometimes bioactive materials with properties similar to those of bone. Nano-composites made of biocompatible polymers and bioactive inorganic nano particles such as HDPE/HA have attracted attention as permanent bone substitutes due to their excellent mechanical properties and biocompatibility. METHOD: The HDPE/HA nano-composite is prepared using melt blending at different HA loading ratios. For evaluation of the degradation by radiation, gamma rays of 35 kGy, and 70 kGy were used to irradiate the samples at room temperature in vacuum. The effects of accelerated ageing after gamma irradiation on morphological, mechanical and thermal properties of HDPE/HA nano-composites were measured. RESULTS: In Vitro test results showed that the HDPE and all HDPE/HA nano-composites do not exhibit any cytotoxicity to WISH cell line. The results also indicated that the tensile properties of HDPE/HA nano-composite increased with increasing the HA content except fracture strain decreased. The dynamic mechanical analysis (DMA) results showed that the storage and loss moduli increased with increasing the HA ratio and the testing frequency. Finally, it is remarked that all properties of HDPE/HA is dependent on the irradiation dose and accelerated aging. CONCLUSION: Based on the experimental results, it is found that the addition of 10%, 20% and 30% HA increases the HDPE stiffness by 23%, 44 and 59% respectively. At the same time, the G' increased from 2.25E11 MPa for neat HDPE to 4.7E11 MPa when 30% HA was added to the polymer matrix. Also, significant improvements in these properties have been observed due to irradiation. Finally, the overall properties of HDPE and its nano-composite properties significantly decreased due to aging and should be taken into consideration in the design of bone substitutes. It is attributed that the developed HDPE/HA nano-composites could be a good alternative material for bone tissue regeneration due to their acceptable properties.