Protein Antigenic Sites Research Articles

Whole-Genome Sequencing and Genetic Diversity of Human Respiratory Syncytial Virus in Patients with Influenza-like Illness in Sicily (Italy) from 2017 to 2023.

Monitoring the genetic variability of human respiratory syncytial virus (hRSV) is of paramount importance, especially for the potential implication of key antigenic mutations on the emergence of immune escape variants. Thus, to describe the genetic diversity and evolutionary dynamics of hRSV circulating in Sicily (Italy), a total of 153 hRSV whole-genome sequences collected from 770 hRSV-positive subjects between 2017 and 2023, before the introduction of expanded immunization programs into the population, were investigated. The phylogenetic analyses indicated that the genotypes GA.2.3.5 (ON1) for hRSV-A and GB.5.0.5a (BA9) for hRSV-B co-circulated in our region. Amino acid (AA) substitutions in the surface and internal proteins were evaluated, including the F protein antigenic sites, as the major targets of immunoprophylactic monoclonal antibodies and vaccines. Overall, the proportion of AA changes ranged between 1.5% and 22.6% among hRSV-A, whereas hRSV-B varied in the range 0.8-16.9%; the latter was more polymorphic than hRSV-A within the key antigenic sites. No AA substitutions were found at site III of both subgroups. Although several non-synonymous mutations were found, none of the polymorphisms known to potentially affect the efficacy of current preventive measures were documented. These findings provide new insights into the global hRSV molecular epidemiology and highlight the importance of defining a baseline genomic picture to monitor for future changes that might be induced by the selective pressures of immunological preventive measures, which will soon become widely available.

Resurgence of respiratory syncytial virus with dominance of RSV-B during the 2022-2023 season.

Respiratory syncytial virus (RSV) is a common cause of upper and lower respiratory tract infections. This study aimed to explore the prevalence of respiratory syncytial virus (RSV) and other respiratory viruses in Bulgaria, characterize the genetic diversity of RSV strains, and perform amino acid sequence analyses of RSV surface and internal proteins. Clinical and epidemiological data and nasopharyngeal swabs were prospectively collected from patients with acute respiratory infections between October 2020 and May 2023. Real-time PCR for 13 respiratory viruses, whole-genome sequencing, phylogenetic, and amino acid analyses were performed. This study included three epidemic seasons (2020-2021, 2021-2022, and 2022-2023) from week 40 of the previous year to week 20 of the following year. Of the 3,047 patients examined, 1,813 (59.5%) tested positive for at least one viral respiratory pathogen. RSV was the second most detected virus (10.9%) after SARS-CoV-2 (22%). Coinfections between RSV and other respiratory viruses were detected in 68 cases, including 14 with SARS-CoV-2. After two seasons of low circulation, RSV activity increased significantly during the 2022-2023 season. The detection rates of RSV were 3.2, 6.6, and 13.7% in the first, second, and third seasons, respectively. RSV was the most common virus found in children under 5 years old with bronchiolitis (40%) and pneumonia (24.5%). RSV-B drove the 2022-2023 epidemic. Phylogenetic analysis indicated that the sequenced RSV-B strains belonged to the GB5.0.5a and GB5.0.6a genotypes. Amino acid substitutions in the surface and internal proteins, including the F protein antigenic sites were identified compared to the BA prototype strain. This study revealed a strong resurgence of RSV in the autumn of 2022 after the lifting of anti-COVID-19 measures, the leading role of RSV as a causative agent of serious respiratory illnesses in early childhood, and relatively low genetic diversity in circulating RSV strains.

Genetic characterization of pigeon- origin avian avulavirus-1 reveals unique substitutions in F and HN proteins.

Since decades, Newcastle disease (ND) has become endemic in the poultry population of the Indian subcontinent. ND is a highly contagious disease of poultry and other avian species. However, the genetic nature of ND viruses circulating in the rock pigeons is unraveled. The present investigation is a part of Newcastle disease virus (NDV) surveillance in wild birds. Two velogenic NDV strains could be isolated from apparently healthy rock pigeons, thus establishing the status of carrier/reservoir host. The fusion protein cleavage site in the fusion protein has multiple basic amino acid (RRRKRF) motifs similar to velogenic isolates. Phylogenetic analysis based on complete fusion gene sequences confirmed that the isolates belong to NDV sub genotype XIII 2.2. Further analysis revealed several amino acid substitutions in the hypervariable region, heptad repeat regions and neutralizing epitopes of the fusion protein and heptad repeat regions and antigenic sites of the hemagglutinin-neuraminidase (HN) protein that are critical for fusion. A unique D170A substitution in the neutralizing epitope is identified that is critical for structure and function of the fusion protein. Mutations within the virulence determinants including fusion (F) and HN, elucidate continuous evolution of the viruses among the rock pigeons. Accidental spillover of these mutated viruses into commercial poultry operations may result in disease outbreaks with economic breakdown.

The Respiratory Syncytial Virus (RSV) G Protein Enhances the Immune Responses to the RSV F Protein in an Enveloped Virus-Like Particle Vaccine Candidate.

Respiratory syncytial virus (RSV) is a serious human respiratory pathogen, but no RSV vaccine has been licensed. Many vaccine candidates are focused on the viral F protein since the F protein is more conserved than the viral G protein across RSV strains and serotypes; thus, the F protein is thought more likely to induce a broader range of protection from infection. However, it is the G protein that binds the likely receptor, CX3CR1, in lung ciliated epithelial cells, raising the question of the importance of the G protein in vaccine candidates. Using virus-like particle (VLP) vaccine candidates, we have directly compared VLPs containing only the prefusion F protein (pre-F), only the G protein, or both glycoproteins. We report that VLPs containing both glycoproteins bind to anti-F-protein-specific monoclonal antibodies differently than do VLPs containing only the prefusion F protein. In RSV-naive cotton rats, VLPs assembled with only the pre-F protein stimulated extremely weak neutralizing antibody (NAb) titers, as did VLPs assembled with G protein. However, VLPs assembled with both glycoproteins stimulated quite robust neutralizing antibody titers, induced improved protection of the animals from RSV challenge compared to pre-F VLPs, and induced significantly higher levels of antibodies specific for F protein antigenic site 0, site III, and the AM14 binding site than did VLPs containing only the pre-F protein. These results indicate that assembly of pre-F protein with G protein in VLPs further stabilized the prefusion conformation or otherwise altered the conformation of the F protein, increasing the induction of protective antibodies. IMPORTANCE Respiratory syncytial virus (RSV) results in significant disease in infants, young children, and the elderly. Thus, development of an effective vaccine for these populations is a priority. Most ongoing efforts in RSV vaccine development have focused on the viral fusion (F) protein; however, the importance of the inclusion of G in vaccine candidates is unclear. Here, using virus-like particles (VLPs) assembled with only the F protein, only the G protein, or both glycoproteins, we show that VLPs assembled with both glycoproteins are a far superior vaccine in a cotton rat model compared with VLPs containing only F protein or only G protein. The results show that the presence of G protein in the VLPs influences the conformation of the F protein and the immune responses to F protein, resulting in significantly higher neutralizing antibody titers and better protection from RSV challenge. These results suggest that inclusion of G protein in a vaccine candidate may improve its effectiveness.

Molecular characterisation of rabies virus detected in livestock animals in the southern part of Egypt during 2018 and 2019

Brain samples were collected from 33 animals of different species, including buffalo, cattle, dog, donkey, fox and wolf, that had been suspected to be infected by rabies virus (RABV) in different geographical regions of Aswan and Luxor governorates in Egypt. The samples were submitted for histopathological examination and the presence of the nucleic acid and antigens of RABV was tested by RT-PCR and indirect fluorescent antibody technique (IFAT), respectively. Sixteen samples were found positive by all the three examinations. Three samples were selected for further study from animals in which the highest virus loads were detected. The partial sequence of the RABV N gene was determined and analysed from the samples of a buffalo, a cow and a donkey. The viruses in the samples were found to share 95-98% and 95-97% nucleotide and amino acid sequence identities, respectively. In comparison to reference sequences, a few amino acid substitutions occurred in the N protein antigenic sites I and IV in the immunodominant epitopes of the viruses detected in the cow and the donkey but not in the one from the buffalo. The phylogenetic analysis revealed that the RABVs sequenced from the samples belonged to genotype 1, Africa-4 clade, and formed two distinct sub-clades within the Egyptian clade. These findings indicate the circulation of RABV among livestock animals in the southern part of Egypt and raise public health concerns. The amino acid changes detected in this work may contribute to the antigenic diversification of RABVs.

Global Molecular Epidemiology of Respiratory Syncytial Virus from the 2017-2018 INFORM-RSV Study.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children, resulting in annual epidemics worldwide. INFORM-RSV is a multiyear clinical study designed to describe the global molecular epidemiology of RSV in children under 5 years of age by monitoring temporal and geographical evolution of current circulating RSV strains, F protein antigenic sites, and their relationships with clinical features of RSV disease. During the pilot season (2017-2018), 410 RSV G-F gene sequences were obtained from 476 RSV-positive nasal samples collected from 8 countries (United Kingdom, Spain, The Netherlands, Finland, Japan, Brazil, South Africa, and Australia). RSV B (all BA9 genotype) predominated over RSV A (all ON1 genotype) globally (69.0% versus 31.0%) and in all countries except South Africa. Geographic clustering patterns highlighted wide transmission and continued evolution with viral spread. Most RSV strains were from infants of <1 year of age (81.2%), males (56.3%), and patients hospitalized for >24 h (70.5%), with no differences in subtype distribution. Compared to 2013 reference sequences, variations at F protein antigenic sites were observed for both RSV A and B strains, with high-frequency polymorphisms at antigenic site Ø (I206M/Q209R) and site V (L172Q/S173L/K191R) in RSV B strains. The INFORM-RSV 2017-2018 pilot season establishes an important molecular baseline of RSV strain distribution and sequence variability with which to track the emergence of new strains and provide an early warning system of neutralization escape variants that may impact transmission or the effectiveness of vaccines and MAbs under development.

Characterization of human respiratory syncytial virus (RSV) isolated from HIV-exposed-uninfected and HIV-unexposed infants in South Africa during 2015-2017.

BackgroundRSV is a leading cause of lower respiratory tract infection in infants. Monitoring RSV glycoprotein sequences is critical for understanding RSV epidemiology and viral antigenicity in the effort to develop anti‐RSV prophylactics and therapeutics.ObjectivesThe objective is to characterize the circulating RSV strains collected from infants in South Africa during 2015‐2017.MethodsA subset of 150 RSV‐positive samples obtained in South Africa from HIV‐unexposed and HIV‐exposed‐uninfected infants from 2015 to 2017, were selected for high‐throughput next‐generation sequencing of the RSV F and G glycoprotein genes. The RSV G and F sequences were analyzed by a bioinformatic pipeline and compared to the USA samples from the same three‐year period.ResultsBoth RSV A and RSV B co‐circulated in South Africa during 2015‐2017, with a shift from RSV A (58%‐61% in 2015‐2016) to RSV B (69%) in 2017. RSV A ON1 and RSV B BA9 genotypes emerged as the most prevalent genotypes in 2017. Variations at the F protein antigenic sites were observed for both RSV A and B strains, with dominant changes (L172Q/S173L) at antigenic site V observed in RSV B strains. RSV A and B F protein sequences from South Africa were very similar to the USA isolates except for a higher rate of RSV A NA1 and RSV B BA10 genotypes in South Africa.ConclusionRSV G and F genes continue to evolve and exhibit both local and global circulation patterns in South Africa, supporting the need for continued national surveillance.

Conserved antigenic sites between MERS-CoV and Bat-coronavirus are revealed through sequence analysis.

BackgroundMERS-CoV is a newly emerged human coronavirus reported closely related with HKU4 and HKU5 Bat coronaviruses. Bat and MERS corona-viruses are structurally related. Therefore, it is of interest to estimate the degree of conserved antigenic sites among them. It is of importance to elucidate the shared antigenic-sites and extent of conservation between them to understand the evolutionary dynamics of MERS-CoV.ResultsMultiple sequence alignment of the spike (S), membrane (M), enveloped (E) and nucleocapsid (N) proteins was employed to identify the sequence conservation among MERS and Bat (HKU4, HKU5) coronaviruses. We used various in silico tools to predict the conserved antigenic sites. We found that MERS-CoV shared 30 % of its S protein antigenic sites with HKU4 and 70 % with HKU5 bat-CoV. Whereas 100 % of its E, M and N protein’s antigenic sites are found to be conserved with those in HKU4 and HKU5.ConclusionThis sharing suggests that in case of pathogenicity MERS-CoV is more closely related to HKU5 bat-CoV than HKU4 bat-CoV. The conserved epitopes indicates their evolutionary relationship and ancestry of pathogenicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13029-016-0049-7) contains supplementary material, which is available to authorized users.

MOLECULAR AND GENETIC CHARACTERISTICS OF SURFACE AND NONSTRUCTURE PROTEINS OF PANDEMIC INFLUENZA VIRUSES A(H1N1)PDM09 IN 2015-2016 EPIDEMIC SEASON

The aim of the present study was identifying of molecular and genetic changes in hemaglutinin (HA), neuraminidase (NA) and non-structure protein (NS1) genes of pandemic influenza A(H1N1)pdm09 strains, that circulated in Ukraine during 2015-2016 epidemic season. Samples (nasopharyngeal swabs from patients) were analyzed using real-time polymerase chain reaction (RTPCR). Phylogenetic trees were constructed using MEGA 7 software. 3D structures were constructed in Chimera 1.11.2rc software. Viruses were collected in 2015-2016 season fell into genetic group 6B and in two emerging subgroups, 6B.1 and 6B.2 by gene of HA and NA. Subgroups 6B.1 and 6B.2 are defined by the following amino acid substitutions. In the NS1 protein were identified new amino acid substitutions D2E, N48S, and E125D in 2015-2016 epidemic season. Specific changes were observed in HA protein antigenic sites, but viruses saved similarity to vaccine strain. NS1 protein acquired substitution associated with increased virulence of the influenza virus.

Protein conformation in a vaccine matters

Protein conformation in a vaccine matters

Molecular characterization of three ferret badger (Melogale moschata) rabies virus isolates from Jiangxi province, China

Ferret badger (FB) rabies viruses JX09-17(fb), JX09-18 and JX10-37 were isolated from three different regions in Jiangxi province, China, in 2009 and 2010. The complete nucleotide sequence identity between these three isolates was 87-93 %. Compared with the other Chinese rabies virus isolates and vaccine strains, 101 substitutions (53 in JX10-37, 23 in JX09-17(fb) and 25 in JX09-18) in the five structural proteins were observed, and 47 of these substitutions (27 in JX10-37, 14 in JX09-17(fb) and 6 in JX09-18) were unique among lyssaviruses. Amino acid substitutions of S231 and Q333 were noted respectively in the G protein antigenic site I of JX10-37 and site III in JX09-17(fb). Phylogenetic analysis showed that JX09-17(fb) is rooted within the China I lineage, JX09-18 is in China II, and JX10-37 is independent. Evolutionary analysis and comparative sequence data indicate that isolate JX10-37 is a variant virus that diverged from canine rabies viruses around 1933 (range 1886-1963).

Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (Kon) for binding to RSV F protein, while alteration of dissociation rate (Koff) did not significantly affect neutralizing activity. Interestingly, linkage of reduced Kon with reduced potency mirrored the effect of increased Kon found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants.

Molecular characterization of a rabies virus isolate from a rabid dog in Hanzhong District, Shaanxi Province, China

A canine rabies virus, Shaanxi-HZ-6, was isolated in Shaanxi Province, China, in 2009. Its genome has been completely sequenced and found to be closely related to the China I rabies virus strains widely circulating in China. The genomic length was 11,923 base pairs, and the overall organization of the genome was similar to that of other rabies virus isolates. Compared with isolates CQ92 and J, 84 amino acid substitutions (7 in the N gene, 15 in P, 6 in M, 25 in G, 31 in L) were observed in strain Shaanxi-HZ-6. Amino acid substitutions of R264H and V332I were noted in the G protein antigenic site I and site III, respectively. Residue 333 of the G protein, which is considered to be associated with pathogenicity, was Arg in Shaanxi-HZ-6. These and other substitutions may help provide an explanation why the China I lineage strain maintains its prevalence in China.

Bioinformatic analysis of embryo development related small heat shock protein Hsp26 in Artemia species

Artemia embryos can endure extreme temperature, long-term anoxia, desiccation and other wide variety of stressful conditions. How the embryos survive these stresses is a very interesting and unsolved subject. To solve this question we analyzed the nucleotide and deduced protein sequence for Hsp26, a molecular chaperone specific to Artemia embryo development. cDNAs of Hsp26 were sequenced from eight Artemia species and deduced Hsp26 amino acid sequences were analyzed. Computer-assisted analysis indicated that the 5′-untranslated region and all the 3 introns contain many putative cis-acting elements for Hsp26 gene expression during development, including heat shock elements (HSEs), Dfd, dl, CF2-II, Hb and AP-1 binding sites. Secondary structure of the Hsp26 3′-untranslated terminator contains the basic structure basis for transcriptional termination. Hsp26 shares sequence similarity with sHSPs (small heat shock protein) from other organisms. The physico-chemical properties of the deduced protein, such as theoretical molecular weight, protein extinction coefficient, isoelectric point and antigenic sites were also obtained. One seven-peptide nuclear localization signals (NLS) “PFRRRMM” was found, which suggested that the Hsp26 protein was hypothesized to be located inside the nucleus. The numbers of phosphorylation sites of serine, threonine and tyrosine and kinase specific phosphorylation sites are also located in Hsp26 protein sequence. These studies will help us achieve a better understanding of Hsp26 and broad implications for sHSPs function in crustacean embryo development.

Genetic Variation Analysis of TGEV Spike Protein Antigenic Sites

Genetic Variation Analysis of TGEV Spike Protein Antigenic Sites

Variability of hemagglutinin-neuraminidase and nucleocapsid protein of vaccine and wild-type mumps virus strains

The mumps virus (MuV) molecular evolution is characterized by the co-circulation of numerous distinct strains. Standardized phylogenetic analyses based on the nucleotide sequences of the SH gene are important for mumps surveillance, but lack the information regarding antigenic properties. So far, the location of antigenic epitopes has been determined for two MuV proteins, the hemagglutinin-neuraminidase (HN) and the nucleocapsid (N) protein. We performed multiple sequence comparisons of putative HN and N protein sequences in order to describe their diversity and plasticity, and to determine the level of similarity between vaccine and wild-type strains. The results of full-length HN or N protein phylogeny showed that MuV strains form a number of differing clades which are in concordance with grouping obtained by standard MuV genotyping. When vaccine strains are compared to all wild-type strains, the highest mean percentage of amino acid differences in both HN and N protein analysis was found for Jeryl Lynn 5 and Jeryl Lynn 2 strains while the lowest value was obtained for Leningrad-3 and L-Zagreb strains. When only 3 antigenic regions of the HN protein, comprising 45 amino acids in total, were investigated, the diversity is considerably diminished: 51.5% of all putative HN proteins show identical sequences (including those of vaccine strains L-Zagreb, Leningrad-3, Hoshino and Urabe). Another 26.5% proteins (including Miyahara vaccine strain) differ in only one amino acid, while the others differ in two to five amino acids from the most common sequence. Jeryl Lynn 2 and Jeryl Lynn 5 strains differ in four amino acids each. N protein antigenic sites have been mapped within its hypervariable C-terminus. Our results indicate that there might be genotype-specific amino acids residing in this antigenic region. The results of our study present the background information for investigations of MuV heterogeneity and antigenic diversity.

Detecting Clostridium botulinum

To the Editor: In the October 2005 issue of Emerging Infectious Diseases, Song et al. described a fiber-optic, microsphere-based, high-density array composed of 18 species-specific probe microsensors, used to identify biological warfare agents, including Clostridium botulinum (1). Although the researchers used multiple probes for C. botulinum, we doubt that this approach is suitable for this organism. C. botulinum comprises a heterogenous group of subspecies that produce botulinum neurotoxin (BoNT); identification and characterization usually rely on animal testing that focuses on antigenetically distinct toxins (2). Although strains of C. botulinum that do not produce toxins are sometimes isolated from wound infections not related to botulism, some strains of C. butyricum and C. baratii are also able to produce BoNTs. The mouse bioassay is currently the accepted method for detecting BoNT. In this assay, mice that receive an intraperitoneal injection containing a sample with more than a minimum lethal dose show symptoms of botulinum intoxication and die. ELISAs, which recognize protein antigenic sites, are still less sensitive than the mouse bioassay (3). Because the mouse bioassay requires euthanizing many animals, and results are not available for several hours, new diagnostic methods are needed. For C. botulinum, an organism widely dispersed in the environment, DNA-based methods may not provide the ultimate solution. Rapid methods to detect and differentiate active BoNTs, such as the rapid, mass spectrometry-based, functional method, are promising candidates to substitute for animal testing in the near future (4).

Immunological studies of sheep brain keratan sulphate proteoglycans

Recently, we reported the isolation and partial characterization of keratan sulphate (KS) from sheep brain. In this study, a panel of monoclonal antibodies (Mab) recognizing epitopes within KS chains and core proteins of KS-containing proteoglycans were used to detect, by immunoblotting, antigenically related molecules extracted from cerebrum, cerebellum and brainstem, respectively. Although the intensity of labelling varied with each of the antibodies, the brain KSPGs were recognized by all the monoclonals used, confirming the presence of KS side chains, which react with the Mabs: 5-D-4, EFG-11, EFG-4, I22, as also the presence of KSPGs related to phosphacan-KS (3H1 proteoglycan). Extracts of all the three brain areas could bind both anti-KS and anti-core protein Mabs, as also anti-HNK-1 monoclonal antibody. Binding was sensitive to keratanases degradation in the cerebrum and brainstem except cerebellum where the presence of a large molecular size hybrid CS/KSPG bearing KS chains partially resistant to keratanases was identified. This population reacts only with 5-D-4, EFG-11 and EFG-4 antibodies. Furthermore, the presence of HNK-1 epitope in CSPGs was detected in the cerebellum and brainstem. In contrast, in the cerebrum the coexistence of HNK-1 epitope and KS in KSPGs was identified. These data suggest that the KSs of sheep brain are part of proteoglycans containing protein and KS antigenic sites related to those of corneal and cartilage KSPG, as also of the brain proteoglycan phosphacan-KS.

Heterologous expression and topography of the main intrinsic protein (MIP) from rat lens

Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88). Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites. Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography. The N- and C-termini were intracellular in both expression systems, and both NPA boxes were found to be extracellular. These results show that residues around the NPA1 box can influence the folding of the MIP in the membrane, and provide structural evidence for the poor water transport properties of MIP, as the NPA boxes lie outside the plane of the membrane.

A Rationally Designed Synthetic Peptide Mimic of a Discontinuous Viral Antigenic Site Elicits Neutralizing Antibodies

A Rationally Designed Synthetic Peptide Mimic of a Discontinuous Viral Antigenic Site Elicits Neutralizing Antibodies