Antigenic Sites Research Articles

Identification and characterization of parvalbumin-like protein in Trichophyton violaceum

Parvalbumins play crucial physiological roles in neuromuscular systems of vertebrates, such as cell-cycle, development of neurons, contraction of muscles, and regulation of intracellular calcium. To perform these neuromuscular functions, parvalbumin may be in associated with other proteins including calbindin, carbonic anhydrase, and cytochrome oxidase. Humans may show an IgE-specific hypersensitivity to parvalbumins after consumption of some distinct fish species. While this protein is abundant in fish muscles, literature review of publications related to fish parvalbumins, do not point to the presence of parvalbumins in eukaryotic microbes. In this study, we propose that distantly related parvalbumins may be found in some non-fish species. Bioinformatics studies such as multiple sequence alignment (MSA), phylogenetic analysis as well as molecular-based experiments indicate that, at least two parvalbumins sequences (UniProt IDs: A0A178F775 and A0A178F7E4) with EF-hand domains and Ca2+-binding sites could be identified in Trichophyton violaceum, a pathogenic fungal species.It was determined that both genes consisted of a single exon and encoded for parvalbumin proteins possessing conserved amino acid motifs. Antigenicity prediction revealed antigenic sites located in both sides of the Ca2+-binding site of the first EF-hand domain. Our phylogenetic analysis revealed that one of parvalbumins (UniProt ID: 0A178F775) can be evolved to other parvalbumins in T. violaceum (UniProt ID: A0A178F7E4) and fish species through evolutionary phenomenon. To confirm our in-silico findings, we designed three primer pairs to detect one of the T. violaceum parvalbumins (UniProt ID: A0A178F7E4) by polymerase chain reaction (PCR); one primer pair showed a strong and specific band in agarose gel electrophoresis. To evaluate the specificity of the method, the primers were tested on extracted DNA from Trichophyton rubrum and T. mentagrophytes. The results demonstrated that the evaluated parvalbumin gene (UniProt ID: A0A178F7E4) was T. violaceum-specific and this pathogenic fungus can be differentiated from T. rubrum and T. mentagrophytes through identification of parvalbumin genes. Further studies are necessary to unravel the biochemical and physiological functions of parvalbumins in T. violaceum.

Epidemic of influenza A(H1N1)pdm09 analyzed by full genome sequences and the first case of oseltamivir-resistant strain in Myanmar 2017.

A community outbreak of human influenza A(H1N1)pdm09 virus strains was observed in Myanmar in 2017. We investigated the circulation patterns, antigenicity, and drug resistance of 2017 influenza A(H1N1)pdm09 viruses from Myanmar and characterized the full genome of influenza virus strains in Myanmar from in-patients and out-patients to assess the pathogenicity of the viruses. Nasopharyngeal swabs were collected from out-patients and in-patients with acute respiratory tract infections in Yangon and Pyinmana City in Myanmar during January-December 2017. A total of 215 out-patients and 18 in-patients infected with A(H1N1)pdm09 were detected by virus isolation and real-time RT-PCR. Among the positive patients, 90.6% were less than 14 years old. Hemagglutination inhibition (HI) antibody titers against A(H1N1)pdm09 viruses in Myanmar were similar to the recommended Japanese influenza vaccine strain for 2017-2018 seasons (A/Singapore/GP1908/2015) and WHO recommended 2017 southern hemisphere vaccine component (A/Michigan/45/2015). Phylogenetic analysis of the hemagglutinin sequence showed that the Myanmar strains belonged to the genetic subclade 6B.1, possessing mutations of S162N and S164T at potential antigenic sites. However, the amino acid mutation at position 222, which may enhance the severity of disease and mortality, was not found. One case with no prior history of oseltamivir treatment possessed H275Y mutated virus in neuraminidase (NA), which confers resistance to oseltamivir and peramivir with elevated IC50 values. The full genome sequence of Myanmar strains showed no difference between samples from in-patients and out-patients, suggesting no additional viral mutations associated with patient severity. Several amino acid changes were observed in PB2, PB1, and M2 of Myanmar strains when compared to the vaccine strain and other Asian strains. However, no mutations associated with pathogenicity were found in the Myanmar strains, suggesting that viral factors cannot explain the underlying reasons of the massive outbreak in Myanmar. This study reported the first detection of an oseltamivir-resistant influenza virus in Myanmar, highlighting the importance of continuous antiviral monitoring and genetic characterization of the influenza virus in Myanmar.

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.

GENOME COMPARATIVE OF EDWARDSIELLA REVEALS POTENTIAL DEVELOPMENT OF SUSTAINABLE PROPHYLAXES

Aquatic diseases caused by the massive wealth of pathogenic bacteria pose major challenges to the development of a sustainable bio-control method, such as antimicrobial measures and vaccine strategies. Recent advances in genome sequencing technology have revolutionized the field of pathogenic pan-genomics and have also influenced disease management in aqua farms. In this study, Edwardsiella strains were differentially classified into four species by a phylogenomics construction based on the pan-genome analysis. Edwardsiella species were correctly classified by pan-genome analysis (core gene, dispensable gene, singleton gene) of 15 complete genomes. Based on the presence of the gene repertoires, gene encoding extracellular protein, outer membrane protein, adhesion ability and antigenic sites, 9 genes (E. ictaluri), 13 genes (E. anguilarium), 9 genes (E. piscicida), 12 genes (E. tadar), and 14 genes (all species) screened from core-gene of 2686, 2673, 2877, 2920, and 1957 gene, respectively have potential in developing reverse vaccinology strategy to the prevention of Edwarsiellosis. The in-silico analysis will also help to optimize the time and improve the cross-serotype reaction of vaccines in farmed fish. The RV research implementing pan-genome analysis will be a strategy that is applicable to pathogens in both aquatic and terrestrial animals.

Interplay between RNA-binding protein HuR and Nox4 as a novel therapeutic target in diabetic kidney disease

ObjectiveGlomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology. MethodsWe studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice. ResultsWe demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3′ untranslated region (3′-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of invitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3′-UTR in response to HG. The invivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function. ConclusionsWe established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.

N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies.

Vaccination is the most effective way to prevent influenza virus infections. However, the diversity of antigenically distinct isolates is a challenge for vaccine development. In order to overcome the antigenic variability and improve the protective efficacy of influenza vaccines, our research group has pioneered the development of computationally optimized broadly reactive antigens (COBRA) for hemagglutinin (HA). Two candidate COBRA HA vaccines, P1 and X6, elicited antibodies with differential patterns of hemagglutination inhibition (HAI) activity against a panel of H1N1 influenza viruses. In order to better understand how these HA antigens elicit broadly reactive immune responses, epitopes in the Cb, Sa, or Sb antigenic sites of seasonal-like and pandemic-like wild-type or COBRA HA antigens were exchanged with homologous regions in the COBRA HA proteins to determine which regions and residues were responsible for the elicited antibody profile. Mice were vaccinated with virus-like particles (VLPs) expressing one of the 12 modified HA antigens (designated V1 to V12), COBRA HA antigens, or wild-type HA antigens. The elicited antisera was assessed for hemagglutination inhibition activity against a panel of historical seasonal-like and pandemic-like H1N1 influenza viruses. Primarily, the pattern of glycosylation sites and residues in the Sa antigenic region, around the receptor binding site (RBS), served as signatures for the elicitation of broadly reactive antibodies by these HA immunogens. Mice were vaccinated with VLPs expressing HA antigens that lacked a glycosylation site at residue 144 and a deleted lysine at position 147 residue were more effective at protecting against morbidity and mortality following infection with pandemic-like and seasonal-like H1N1 influenza viruses.IMPORTANCE There is a great need to develop broadly reactive or universal vaccines against influenza viruses. Advanced, next-generation hemagglutinin (HA) head-based vaccines that elicit protective antibodies against H1N1 influenza viruses have been developed. This study focused on understanding the specific amino acids around the receptor binding site (RBS) that were important in elicitation of these broadly reactive antibodies. Specific glycan sites and amino acids located at the tip of the HA molecule enhanced the elicitation of these broadly reactive antibodies. A better understanding of the HA structures around the RBS will lead to more effective HA immunogens.

Genetic Diversity of Human Rotavirus A Among Hospitalized Children Under-5 Years in Lebanon.

Human rotavirus remains a major cause of gastroenteritis worldwide despite the availability of effective vaccines. In this study, we investigated the genetic diversity of rotaviruses circulating in Lebanon. We genetically characterized the VP4 and VP7 genes encoding the outer capsid proteins of 132 rotavirus-associated gastroenteritis specimens, previously identified in hospitalized children (<5 years) from 2011 to 2013 in Lebanon. These included 43 vaccine-breakthrough specimens and the remainder were from non-vaccinated subjects. Phylogenetic analysis of VP4 and VP7 genes revealed distinct clustering compared to the vaccine strains, and several substitutions were identified in the antigenic epitopes of Lebanese specimens. No unique changes were identified in the breakthrough specimens compared to non-breakthroughs that could explain the occurrence of infection in vaccinated children. Further, we report the emergence of a rare P[8] OP354-like strain with a G9 VP7 in Lebanon, possessing high genetic variability in their VP4 compared to vaccine strains. Therefore, human rotavirus strains circulating in Lebanon and globally have accumulated numerous substitutions in their antigenic sites compared to those currently used in the licensed vaccines. The successful spread and continued genetic drift of these strains over time might undermine the effectiveness of the vaccines. The effect of such changes in the antigenic sites on vaccine efficacy remains to be assessed.

Substantial Antigenic Drift in the Hemagglutinin Protein of Swine Influenza A Viruses.

The degree of antigenic drift in swine influenza A viruses (swIAV) has historically been regarded as minimal compared to that of human influenza A virus strains. However, as surveillance activities on swIAV have increased, more isolates have been characterized, revealing a high level of genetic and antigenic differences even within the same swIAV lineage. The objective of this study was to investigate the level of genetic drift in one enzootically infected swine herd over one year. Nasal swabs were collected monthly from sows (n = 4) and piglets (n = 40) in the farrowing unit, and from weaners (n = 20) in the nursery. Virus from 1–4 animals were sequenced per month. Analyses of the sequences revealed that the hemagglutinin (HA) gene was the main target for genetic drift with a substitution rate of 7.6 × 10−3 substitutions/site/year and evidence of positive selection. The majority of the mutations occurred in the globular head of the HA protein and in antigenic sites. The phylogenetic tree of the HA sequences displayed a pectinate typology, where only a single lineage persists and forms the ancestor for subsequent lineages. This was most likely caused by repeated selection of a single immune-escape variant, which subsequently became the founder of the next wave of infections.

Neutralization capacity of highly divergent type 2 vaccine-derived polioviruses from immunodeficient patients

The use of the oral poliovirus vaccine (OPV) in developing countries has reduced the incidence of poliomyelitis by >99% since 1988 and is the primary tool for global polio eradication. Spontaneous reversions of the vaccine virus to a neurovirulent form can impede this effort. In persons with primary B-cell immunodeficiencies, exposure to OPV can result in chronic infection, mutation, and excretion of immunodeficiency-associated vaccine-derived polioviruses, (iVDPVs). These iVDPVs may have the potential for transmission in a susceptible population and cause paralysis. The extent to which sera from OPV recipients are able to neutralize iVDPVs with varying degrees of antigenic site substitutions is investigated here. We tested sera from a population immunized with a combination vaccine schedule (both OPV and inactivated polio vaccine) against a panel of iVDPVs and found that increases in amino acid substitution in the P1 capsid protein resulted in a decrease in the neutralizing capacity of the sera. This study underscores the importance of maintaining high vaccine coverage in areas of OPV use as well as active surveillance of those known to be immunocompromised.

Human Antibody Repertoire following Ebola Virus Infection and Vaccination.

SummaryLimited knowledge exists on the quality of polyclonal antibody response generated following Ebola virus (EBOV) infection compared with vaccination. Polyclonal antibody repertoire in plasma following EBOV infection in survivors was compared with ChAd3-MVA prime-boost human vaccination. Higher antibody binding and affinity to GP was observed in survivors compared with vaccinated plasma that correlated with EBOV neutralization. Surprisingly, a predominant IgM response was generated after prime-boost vaccination, whereas survivors demonstrated IgG-dominant antibody response. EBOV infection induced more diverse antibody epitope repertoire compared with vaccination. A strong binding to antigenic sites in the fusion peptide and another in the highly conserved GP2-HR2 domain was preferentially recognized by EBOV survivors than vaccinated individuals that correlated strongly with EBOV neutralization titers. These findings will help development and evaluation of effective Ebola countermeasures including therapeutics and vaccines.

Nanobodies: The "Magic Bullets" in therapeutics, drug delivery and diagnostics.

Antibodies represent a well-established class of clinical diagnostics for medical applications as well as essential research and biotechnological tools. Although both polyclonal and monoclonal antibodies are indispensable reagents in basic research and diagnostics but both of them have their limitations. Hence, there is urgent need to develop strategies aimed at production of alternative scaffolds and recombinant antibodies of smaller dimensions that could be easily produced, selected and manipulated. Unlike conventional antibodies, members of Camelidae and sharks produce antibodies composed only of heavy chains with small size, high solubility, thermal stability, refolding capacity and good tissue penetration in vivo. The discovery of these naturally occurring antibodies having only heavy-chain in Camelidae family and their further development into small recombinant nanobodies represents an attractive alternative in drug delivery, diagnostics and imaging. Nanobody derivatives are soluble, stable, versatile, have unique refolding capacities, reduced aggregation tendencies and high-target binding capabilities. They can be genetically customized to target enzymes, transmembrane proteins or molecular interactions. Their ability to recognize recessed antigenic sites has been attributed to their smaller size and the ability of the extended CDR3 loop to quickly penetrate into such epitopes. With the advent of molecular engineering and phage display technology, they can be of potential use in molecular imaging, drug delivery and therapeutics for several major diseases. In this review we present the recent advances in nanobodies for modulating immune functions, for targeting cancers, viruses, toxins and microbes as well as their utility as diagnostic and biosensor tools.

Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene.

Influenza A (H1N1) pdm09 virus emerged in North America in 2009 and has been established as a seasonal strain in humans. After an antigenic stasis of about six years, new antigenically distinct variants of the virus emerged globally in 2016 necessitating a change in the vaccine formulation for the first time in 2017. Herein, we analyzed thirty-eight HA sequences of influenza A (H1N1) pdm09 strains isolated in Kenya during 2015-2018 seasons, to evaluate their antigenic and molecular properties based on the HA1 sub-unit. Our analyses revealed that the A (H1N1) pdm09 strains that circulated in Kenya during this period belonged to genetic clade 6B, subclade 6B.1 and 6B.2. The Kenyan 2015 and 2016 isolates differed from the vaccine strain A/California/07/2009 at nine and fourteen antigenic sites in the HA1 respectively. Further, those isolated in 2017 and 2018 correspondingly varied from A/Michigan/45/2015 vaccine strain at three and fifteen antigenic sites. The predicted vaccine efficacy of A/California/07/2009 against Kenyan 2015/2016 was estimated to be 32.4% while A/Michigan/45/2015 showed estimated vaccine efficacies of 39.6% - 41.8% and 32.4% - 42.1% against Kenyan 2017 and 2018 strains, respectively. Hemagglutination-inhibition (HAI) assay using ferret post-infection reference antiserum showed that the titers for the Kenyan 2015/2016 isolates were 2-8-fold lower compared to the vaccine strain. Overall, our results suggest the A (H1N1) pdm09 viruses that circulated in Kenya during 2015/2016 influenza seasons were antigenic variants of the recommended vaccine strains, denoting sub-optimal vaccine efficacy. Additionally, data generated point to a swiftly evolving influenza A (H1N1) pdm09 virus in recent post pandemic era, underscoring the need for sustained surveillance coupled with molecular and antigenic analyses, to inform appropriate and timely influenza vaccine update.

Novel Variants of Respiratory Syncytial Virus A ON1 Associated With Increased Clinical Severity of Bronchiolitis.

A study of respiratory syncytial virus-A (RSV A) genotype ON1 genetic variability and clinical severity in infants hospitalized with bronchiolitis over 6 epidemic seasons (2012-2013 to 2017-2018) was carried out. From prospectively enrolled term infants hospitalized for bronchiolitis, samples positive for RSV A ON1 (N = 139) were sequenced in the second half of the G gene. Patients' clinical data were obtained from medical files and each infant was assigned a clinical severity score. ANOVA comparison and adjusted multinomial logistic regression were used to evaluate clinical severity score and clinical parameters. The phylogenetic analysis of 54 strains showed 3 distinct clades; sequences in the last 2 seasons differed from previous seasons. The most divergent and numerous cluster of 2017-2018 strains was characterized by a novel pattern of amino acid changes, some in antigenic sites. Several amino acid changes altered predicted glycosylation sites, with acquisition of around 10 new O-glycosylation sites. Clinical severity of bronchiolitis increased in 2016-2017 and 2017-2018 and changed according to the epidemic seasons only. Amino acid changes in the hypervariable part of G protein may have altered functions and/or changed its immunogenicity, leading to an impact on disease severity.

Genetic characterization of bovine ephemeral fever virus in southern China, 2013-2017.

Bovine ephemeral fever virus (BEFV) can cause bovine ephemeral fever and is an economically important arbovirus of cattle. To expand the knowledge of the molecular epidemiology of BEFV in southern China, the complete surface glycoprotein G gene of BEFV was sequenced from samples collected in five restricted outbreaks from 2013 to 2017, namely 2013ZH, 2014HM, 2015GX, 11082-2016, and qy2017. It was noted that both 2014HM and 11082-2016 were detected in cattle regularly vaccinated with inactivated vaccine. Phylogenetic analysis demonstrated that all five strains grouped into cluster I. However, qy2017 was closer to the BEFV strains identified in Thailand, Japan, and Taiwan after 2000, while 2013ZH, 2014HM, 2015GX, and 11082-2016 were closer to the Chinese strains in 2011 and the Turkey strains in 2012. The analysis of antigenic sites indicated that several amino acid changes occurred between the five strains and the vaccine strain. Importantly, one novel amino acid mutation site was observed in the putative N-linked glycosylation sites of 2013ZH, 2014HM, 2015GX, and 11082-2016. Our study indicated novel genetic characteristics of the newly emerging BEFV strains in southern China and the necessity of updating the component of commercially available inactivated BEFV vaccines in China.

Mutations in foot and mouth disease virus types A and O isolated from vaccinated animals.

Foot and mouth disease (FMD) is responsible for serious economic losses in Egypt. Although vaccination is practised as the main control strategy, failure of vaccination has been reported in many cases, which can be due to a number of factors. Selection of FMD antigenic variants under the immune pressure of partially immunised hosts has been previously recorded. This study was designed to isolate and characterise foot and mouth disease viruses (FMDVs) circulating in Egyptian vaccinated animals. Serotype O, A and Southern African Territories (SAT) 2 FMDVs were detected in different Egyptian governorates during 2015, 2016 and 2017. The successful isolation of 15 FMDVs of the three serotypes is reported in this paper. Phylogenetic analysis based on the viral protein (VP) 1 gene showed that all serotype O isolates had East Africa (EA)-3 topotypes. There was variation in 15-17 amino acids between the serotype O isolates of 2015 and those of 2016 and 2017. The serotype A isolates belonged to the A-Iran-05 lineage, with the exception of one isolate of 2016 which clustered with the African strains of G-IV. Serotype SAT2 FMDV was detected in two samples of 2017 and both were of lineage Alx-12 of topotype VII. The virus neutralisation test using sera raised against vaccine strains confirmed the serotyping of the isolates and determined the antigenic relatedness between the isolates and the currently used vaccine strains. A decrease in the neutralising antibody titre of some serotype O and A isolates could be attributed to mutation in critical amino acids in the neutralising antigenic sites. Hence, this work supports previous studies describing the significance of amino acid substitutions within the antigenic sites of the virus in antibody neutralisation and immune escape.

Uncovering the Anti-Ebola Repertome

Ebolavirus disease is a global threat. In this issue, Khurana etal. reveal the antibody response against all ebolavirus proteins by analyzing longitudinal antibody repertoires of an Ebola survivor from disease onset. Antibodies against VP40 and GP are found to predominate and two protective antigenic sites in GP identified.

Genomic and biological characterization of a pandemic norovirus variant GII.4 Sydney 2012.

Genogroup II, genotype 4 noroviruses (GII.4 NoVs) are a leading cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. In this study, we isolated a GII.4 NoV strain (designated 2015HN08) from a kid presenting with acute gastroenteritis and determined its near-complete genome sequence. We then performed sequence analysis by comparing this strain with the prototypical GII.4 strain. Virus-like particles (VLPs) derived from the major capsid protein (VP1) were expressed by using a recombinant-baculovirus expression system, and monoclonal antibodies (mAbs) were produced to compare changes in antigenic or histo-blood group antigens (HBGAs) binding sites with the previously characterized GII.4 NoV strain (JZ403). The genome of 2015HN08 was 7559 nucleotides (nt) long, excluding the poly(A) tail. Genotyping analysis indicated that this strain was a Sydney 2012 variant. In comparison with the prototype Sydney 2012 strain, there were 74, 35, and 16 differences in nucleotide sequences in ORF1, OFR2, and OFR3, causing 7, 10, and 6 amino acid (aa) changes, respectively. Expression of VP1 led to successful assembly of VLPs, as demonstrated by electron microscopy. Screening of hybridoma cell supernatants with an in vitro VLP-HBGAs binding blockade assay led to the identification of a cell clone 3G10 that exhibited HBGA-blocking effects. This mAb also exhibited blocking effects against JZ403 strain, suggesting maintenance of the antigenic site and/or HBGAs binding sites between the two strains. In summary, we determined the near-complete genome sequence of a GII.4 Sydney 2012 variant and produced an mAb with blocking effects that might be useful in evaluating the evolution of current Sydney 2012 NoV strains.

Linking influenza virus evolution within and between human hosts.

Influenza viruses rapidly diversify within individual human infections. Several recent studies have deep-sequenced clinical influenza infections to identify viral variation within hosts, but it remains unclear how within-host mutations fare at the between-host scale. Here, we compare the genetic variation of H3N2 influenza within and between hosts to link viral evolutionary dynamics across scales. Synonymous sites evolve at similar rates at both scales, indicating that global evolution at these putatively neutral sites results from the accumulation of within-host variation. However, nonsynonymous mutations are depleted between hosts compared to within hosts, suggesting that selection purges many of the protein-altering changes that arise within hosts. The exception is at antigenic sites, where selection detectably favors nonsynonymous mutations at the global scale, but not within hosts. These results suggest that selection against deleterious mutations and selection for antigenic change are the main forces that act on within-host variants of influenza virus as they transmit and circulate between hosts.

Serological evaluation and differentiation of subgroups of “A” and “AB” in healthy blood donor population in Eastern India

Background and Objectives: Weak subgroups of A antigen in A or AB blood group can be differentiated by various immunohematological investigations. These weak phenotypes mainly result from weaker expression of an alternate weak allele present at the ABO loci. Weak subgroups of A antigen either reacts weakly with anti-A antisera such as A3, Ax, and Aendor do not react at all such as Am, Ay, and Ael. In this study, we characterized and differentiated the broad and weak subgroups of A in ABO blood group system through various immunohematological investigations. Methods: The prospective study included 67,954 healthy blood donors. Initial blood grouping and antibody screening of all donor samples were performed using an automated solid-phase assay. Samples showing blood group discrepancy or weaker agglutination were subjected to battery of serological investigation including saliva hemagglutination inhibition test and adsorption-elution procedures. Results: Among 16,034 A group donors, A1were 13,276 (82.8%), A22750 (17.15%), and weak subgroups were 8 (0.05%). Among AB group, A1B donors were 5621 (84.93%), A2B 996 (15.05%), and weak subgroups of AB were 1 (0.015%). The unexpected naturally occurring anti-A1antibodies (1.9%) were of IgM type optimally reactive at 4°C with titer ranging from 1 to 8. Red cells' agglutination with anti-A and anti-AB varied from Wk + to 2+ with or without mixed field agglutination in A3, Ax, and Aendphenotypes. While saliva study revealed specific substances, adsorption-elution demonstrated “A” antigen specificity in different strengths in Am, Ay, and AmB phenotypes. Conclusion: The subgroups of A and AB are attributed to a reduced number of A antigen sites on the red cells. We conclude that differentiating weak subgroups of “A” by serological assays is possible to a great extend with technical expertise; however, molecular genotyping is essential because it confirms the result.

Alpha-fetoprotein is an autoantigen in hepatocellular carcinoma and juvenile Batten disease.

Failure of immune tolerance leads to production of autoantibodies to self-antigens. The repertoire of autoantibodies detected in cancer patients can indicate the presence of autoimmune disease. Alpha-fetoprotein (AFP) autoantibodies have been found in patients withhepatocellular carcinoma (HCC) and in juvenile Batten disease (BD), a neurodegenerative condition involving autoimmunity. Variant conformational forms of AFP together with exposed occult antigenic determinant sites on the AFP polypeptide resemble the features of a disordered proteinwhich can impair central immune tolerance. These aberrant structural protein forms can lead to the persistence of autoantibody production byimmune sensitized B-lymphocytes. Thus, it is not surprisingthat AFP, a self-antigen, can induce autoimmune responses in humans. Herein, we discuss the molecularand antigenic properties of AFP which make it a disordered protein, and its ability to induce autoantibody production to AFP cryptic epitopes in both HHC and BDpatients. Such insights might aid in the future design of AFP-based vaccines and to discovery of novel pathogenic mechanisms of autoimmune diseases which demonstrate the presence of denatured intermediate forms of AFP.