RESUMO
Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine.
Assuntos
Substituição de Aminoácidos , Vacina Antirrábica , Vírus da Raiva , Animais , Células Vero , Chlorocebus aethiops , Vacina Antirrábica/genética , Vacina Antirrábica/imunologia , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Humanos , Raiva/prevenção & controle , Raiva/virologia , Genoma ViralRESUMO
Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.
Assuntos
Substituição de Aminoácidos , Vírus da Raiva , Raiva , Proteínas da Matriz Viral , Virulência , Animais , Camundongos , Encéfalo/virologia , Linhagem Celular , Neurônios/virologia , Neurônios/patologia , Raiva/virologia , Vírus da Raiva/genética , Vírus da Raiva/patogenicidade , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Virulência/genética , Replicação ViralRESUMO
During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.
Assuntos
Cervos , Flavivirus , Metagenômica , Carrapatos , Animais , Metagenômica/métodos , Japão/epidemiologia , Cervos/virologia , Flavivirus/genética , Flavivirus/isolamento & purificação , Flavivirus/classificação , Carrapatos/virologia , Filogenia , Viroma/genética , Vírion/genética , Sus scrofa/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Soroepidemiológicos , Genoma ViralRESUMO
Some lyssaviruses, including the rabies virus (RABV), cause lethal neurological symptoms in humans. However, the efficacy of commercial vaccines has only been evaluated against RABV. To assess cross-reactivity among lyssaviruses, including RABV, sera from rabbits inoculated with human and animal RABV vaccines and polyclonal antibodies from rabbits immunized with expression plasmids of the glycoproteins of all 18 lyssaviruses were prepared, and cross-reactivity was evaluated via virus-neutralization tests using Duvenhage lyssavirus (DUVV), European bat lyssavirus-1 (EBLV-1), Mokola lyssavirus (MOKV), Lagos bat lyssavirus (LBV), and RABV. The sera from rabbits inoculated with RABV vaccines showed cross-reactivity with EBLV-1 and DUVV, both belonging to phylogroup I. However, reactivity with MOKV and LBV in phylogroup II was notably limited or below the detection level. Next, we compared the cross-reactivity of the polyclonal antibodies against all lyssavirus glycoproteins. Polyclonal antibodies had high virus-neutralization titers against the same phylogroup but not different phylogroups. Our findings indicate that a new vaccine should be developed for pre- and post-exposure prophylaxis against lyssaviral infections.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Reações Cruzadas , Glicoproteínas , Lyssavirus , Testes de Neutralização , Animais , Lyssavirus/imunologia , Coelhos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Glicoproteínas/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Humanos , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/prevenção & controleRESUMO
Rabies is a fatal zoonotic, neurological disease caused by rabies lyssavirus (RABV) and other lyssaviruses. In this study, we established novel serological neutralizing tests (NT) based on vesicular stomatitis virus pseudotypes possessing all 18 known lyssavirus glycoproteins. Applying this system to comparative NT against rabbit sera immunized with current RABV vaccines, we showed that the current RABV vaccines fail to elicit sufficient neutralizing antibodies against lyssaviruses other than to those in phylogroup I. Furthermore, comparative NT against rabbit antisera for 18 lyssavirus glycoproteins showed glycoproteins of some lyssaviruses elicited neutralizing antibodies against a broad range of lyssaviruses. This novel testing system will be useful to comprehensively detect antibodies against lyssaviruses and evaluate their cross-reactivities for developing a future broad-protective vaccine.
Assuntos
Lyssavirus , Vacina Antirrábica , Vírus da Raiva , Raiva , Animais , Coelhos , Raiva/veterinária , Anticorpos Antivirais , Pseudotipagem Viral/veterinária , Anticorpos Neutralizantes , Glicoproteínas , ZoonosesRESUMO
Nipah virus (NiV) is a highly pathogenic zoonotic virus that causes severe encephalitis and respiratory diseases and has a high mortality rate in humans (>40%). Epidemiological studies on various fruit bat species, which are natural reservoirs of the virus, have shown that NiV is widely distributed throughout Southeast Asia. Therefore, there is an urgent need to develop effective NiV vaccines. In this study, we generated recombinant vaccinia viruses expressing the NiV glycoprotein (G) or fusion (F) protein using the LC16m8 strain, and examined their antigenicity and ability to induce immunity. Neutralizing antibodies against NiV were successfully induced in hamsters inoculated with LC16m8 expressing NiV G or F, and the antibody titers were higher than those induced by other vaccinia virus vectors previously reported to prevent lethal NiV infection. These findings indicate that the LC16m8-based vaccine format has superior features as a proliferative vaccine compared with other poxvirus-based vaccines. Moreover, the data collected over the course of antibody elevation during three rounds of vaccination in hamsters provide an important basis for the clinical use of vaccinia virus-based vaccines against NiV disease. Trial Registration: NCT05398796.
Assuntos
Infecções por Henipavirus , Vírus Nipah , Vacinas Virais , Animais , Cricetinae , Humanos , Vaccinia virus/genética , Vírus Nipah/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Vacinas Virais/genética , Vacinas Sintéticas/genética , Infecções por Henipavirus/prevenção & controleRESUMO
Background: The immune profile against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by Omicron in individuals with various immune histories. Methods: The neutralization susceptibility of the variants, including Omicron and their ancestors, was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections of Alpha/Delta with multiple time intervals following vaccination. Findings: Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against Omicron was induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies. Conclusions: Immune histories with breakthrough infections can overcome the resistance to infection by Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against Omicron and future variants. Funding: This study was supported by grants from the Japan Agency for Medical Research and Development (AMED).
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Vacina BNT162 , Vacinas contra COVID-19 , Humanos , Complicações Pós-Operatórias , VacinaçãoRESUMO
Rabies is a highly neurotropic disease caused by rabies lyssavirus (RABV). Human rabies vaccines exist for pre- and postexposure prophylaxis; however, after clinical symptoms appear, the disease has an â¼100% mortality rate with no effective treatments available. In our previous study, mouse neuroblastoma cells transfected with a plasmid coding one clone of a single-chain variable fragment (scFv), scFv-P19, against RABV phosphoprotein (RABV-P) derived from an scFv phage-display library, before infection, exhibited reduced viral propagation after infection with the RABV-fixed strain, CVS11. In this study, we conducted epitope mapping of scFv-P19 through indirect fluorescent assay and Western blotting analysis against full-length and N- or C-terminal truncated RABV-P. Our results suggest that scFv-P19 targets a portion containing amino acids 47-52 at the N-terminus, which partially overlaps with the N-terminal nuclear export sequences. This provides insights into the underlying mechanism associated with inhibition of RABV by scFv-P19, while allowing for the design of additional scFv-based therapeutic studies for RABV by integrating appropriate delivery and application systems. Furthermore, the results of this study suggest that scFv-P19 may serve as an effective tool for investigating nuclear trafficking of RABV-P to explore the roles of RABV-P isoforms in rabies pathogenesis.
Assuntos
Vírus da Raiva , Raiva , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais/farmacologia , Mapeamento de Epitopos , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/farmacologia , Vírus da Raiva/metabolismo , Anticorpos de Cadeia Única/genéticaAssuntos
Mordeduras e Picadas , Vírus da Raiva , Raiva , Humanos , Japão , Raiva/prevenção & controleRESUMO
BACKGROUND: Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe encephalitis and respiratory disease with a high mortality rate in humans. During large outbreaks of the viral disease, serological testing of serum samples could be a useful diagnostic tool, which could provide information on not only the diagnosis of NiV disease but also the history of an individual with previous exposure to the virus, thereby supporting disease control. Therefore, an efficient method for the inactivation of NiV in serum samples is required for serological diagnosis. METHODS: We determined the optimal conditions for the inactivation of NiV infectivity in human serum using heating and UV treatment. The inactivation method comprised UV irradiation with a cover of aluminum foil for 30 min and heating at 56 °C for 30 min. RESULTS: With an optimized protocol for virus inactivation, NiV infectivity in serum samples (containing 6.0 × 105 TCID50) was completely inactivated. CONCLUSIONS: We developed a recommended protocol for the effective inactivation of NiV. This protocol would enable a regional or local laboratory to safely transport or process samples, including NiV, for serological testing in its biosafety level-2 facility.
Assuntos
Temperatura Alta , Viabilidade Microbiana/efeitos da radiação , Vírus Nipah/efeitos da radiação , Raios Ultravioleta , Virologia/métodos , Inativação de Vírus/efeitos da radiação , Animais , Chlorocebus aethiops , Infecções por Henipavirus/sangue , Infecções por Henipavirus/virologia , Humanos , Vírus Nipah/fisiologia , Pesquisa , Células VeroRESUMO
BACKGROUND: The complete genome sequences of 44 Bacillus cereus group isolates collected from diverse sources in Japan were analyzed to determine their genetic backgrounds and diversity levels in Japan. Multilocus sequence typing (MLST) and core-genome single-nucleotide polymorphism (SNP) typing data from whole-genome sequences were analyzed to determine genetic diversity levels. Virulence-associated gene profiles were also used to evaluate the genetic backgrounds and relationships among the isolates. RESULTS: The 44 B. cereus group isolates, including soil- and animal-derived isolates and isolates recovered from hospitalized patients and food poisoning cases, were genotyped by MLST and core-genome SNP typing. Genetic variation among the isolates was identified by the MLST and core-genome SNP phylogeny comparison against reference strains from countries outside of Japan. Exploratory principal component analysis and nonmetric multidimensional scaling (NMDS) analyses were used to assess the genetic similarities among the isolates using gene presence and absence information and isolate origins as the metadata. A significant correlation was seen between the principal components and the presence of genes encoding hemolysin BL and emetic genetic determinants in B. cereus, and the capsule proteins in B. anthracis. NMDS showed that the cluster of soil isolates overlapped with the cluster comprising animal-derived and clinical isolates. CONCLUSIONS: Molecular and epidemiological analyses of B. cereus group isolates in Japan suggest that the soil- and animal-derived bacteria from our study are not a significant risk to human health. However, because several of the clinical isolates share close genetic relationships with the environmental isolates, both molecular and epidemiological surveillance studies could be used effectively to estimate virulence in these important pathogens.
Assuntos
Bacillus cereus/genética , Bacillus cereus/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Microbiologia do Solo , Animais , Bacillus anthracis/genética , Bacillus cereus/classificação , Bacillus cereus/isolamento & purificação , Técnicas de Tipagem Bacteriana , Doenças Transmitidas por Alimentos/microbiologia , Variação Genética , Genótipo , Infecções por Bactérias Gram-Positivas/epidemiologia , Hospitalização , Humanos , Japão/epidemiologia , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Virulência , Sequenciamento Completo do GenomaRESUMO
A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries.
Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Técnica Indireta de Fluorescência para Anticorpo/métodos , Infecções por Henipavirus/diagnóstico , Imunoglobulina M/sangue , Animais , Proteínas do Capsídeo/imunologia , Células HeLa , Infecções por Henipavirus/sangue , Infecções por Henipavirus/imunologia , Humanos , Macaca/imunologia , Macaca/virologia , Vírus Nipah , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodosRESUMO
Intracellular antibodies (intrabodies) are expected to function as therapeutics as well as tools for elucidating in vivo function of proteins. In this study, we propose a novel intrabody selection method in the cytosol of mammalian cells by utilizing a growth signal, induced by the interaction of the target antigen and an scFv-c-kit growth sensor. Here, we challenge this method to select specific intrabodies against rabies virus nucleoprotein (RV-N) for the first time. As a result, we successfully select antigen-specific intrabodies from a naïve synthetic library using phage panning followed by our growth sensor-based intracellular selection method, demonstrating the feasibility of the method. Additionally, we succeed in improving the response of the growth sensor by re-engineering the linker region of its construction. Collectively, the described selection method utilizing a growth sensor may become a highly efficient platform for selection of functional intrabodies in the future.
Assuntos
Anticorpos/genética , Citosol/metabolismo , Engenharia de Proteínas/métodos , Animais , Anticorpos/imunologia , Nucleoproteínas/imunologia , Biblioteca de Peptídeos , Vírus da RaivaRESUMO
A versatile strategy to inhibit protein functions in the cytoplasmic environment is eagerly anticipated for drug discovery. In this study, we demonstrate a novel system to directly select functional intrabodies from a library in the mammalian cytoplasm. In this system, a target homo-oligomeric antigen is expressed together with a single-chain Fv (scFv) library that is linked to the cytoplasmic domain of a receptor tyrosine kinase (RTK) in the cytoplasm of murine interleukin-3 (IL-3)-dependent cells. As the tyrosine kinase is activated by dimerization, only scFv-RTK clones that can bind to the target antigen would be oligomerized and transduce a growth signal under the IL-3-deprived condition, which leads to selection of functional intrabodies. To demonstrate this system, we used rabies virus phosphoprotein (RV-P) that forms dimers in the cytoplasm as a target antigen. As a result, functional intrabodies were selected using our system from a naïve scFv library as well as from a pre-selected anti-RV-P library generated by phage display. This system may be applied for screening intrabodies that can prevent progression of various severe diseases.
Assuntos
Antígenos/metabolismo , Engenharia de Proteínas/métodos , Proteínas Tirosina Quinases/metabolismo , Anticorpos de Cadeia Única/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Citoplasma/metabolismo , Humanos , Interleucina-3/farmacologia , Camundongos , Biblioteca de Peptídeos , Multimerização Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Anticorpos de Cadeia Única/genéticaRESUMO
We identified novel viruses in feces from cattle with diarrhea collected in 2009 in Hokkaido Prefecture, Japan, by using a metagenomics approach and determined the (near) complete sequences of the virus. Sequence analyses revealed that they had a standard picornavirus genome organization, i.e. 5' untranslated region (UTR) - L- P1 (VP4- VP3- VP2- VP1) - P2 (2A- 2B- 2C) - P3 (3A- 3B- 3C-3D) - 3'UTR- poly(A). They are closely related to other unclassified Chinese picornaviruses; bat picornaviruses group 1-3, feline picornavirus, and canine picornavirus, sharing 45.4-51.4% (P1), 38.0-44.9% (P2), and 49.6-53.3% (P3) amino acid identities, respectively. The phylogenetic analyses and detailed genome characterization showed that they, together with the unclassified Chinese picornaviruses, grouped as a cluster for the P1, 2C, 3CD and VP1 coding regions. These viruses had conserved features (e.g. predicted protein cleavage sites, presence of a leader protein, 2A, 2C, 3C, and 3D functional domains), suggesting they have a common ancestor. Reverse-transcription-PCR assays, using specific primers designed from the 5'UTR sequence of these viruses, showed that 23.0% (20/87) of fecal samples from cattle with diarrhea were positive, indicating the prevalence of these picornavirus in the Japanese cattle population in Hokkaido Prefecture. However, further studies are needed to investigate the pathogenic potential and etiological role of these viruses in cattle.
Assuntos
Doenças dos Bovinos/virologia , Genoma Viral , Infecções por Picornaviridae/veterinária , Picornaviridae/classificação , Picornaviridae/isolamento & purificação , RNA Viral/genética , Análise de Sequência de DNA , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Análise por Conglomerados , Diarreia/epidemiologia , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Ordem dos Genes , Genes Virais , Japão/epidemiologia , Dados de Sequência Molecular , Filogenia , Picornaviridae/genética , Infecções por Picornaviridae/virologia , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de SequênciaRESUMO
We report the draft genome sequences of Bacillus anthracis strains Shikan-NIID, 52-40-NIAH, and 44-NIAH stored in Japan and belonging to the A3 cluster.
RESUMO
In this study, G proteins of the rabies virus (RABV) Kyoto strain were detected in the cytoplasm but not distributed at the cell membrane of mouse neuroblastoma (MNA) cells. G proteins of CVS-26 were detected in both the cell membrane and perinuclear space of MNA cells. We found that N-glycosylation of street RABV G protein by the insertion of the sequon Asn(204) induced the transfer of RABV G proteins to the cell surface membrane. Fixed RABV budding from the plasma membrane has been found to depend not only on G protein but also on other structural proteins such as M protein. However, the differing N-glycosylation of G protein could be associated with the distinct budding and antigenic features of RABV in street and fixed viruses. Our study of the association of N-glycan of G protein at Asn(204) with the transport of RABV G protein to the cell surface membrane contributes to the understanding of the evolution of fixed virus from street virus, which in turn would help for determine the mechanism underlying RABV budding and enhanced host immune responses.
Assuntos
Antígenos Virais/química , Antígenos Virais/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Transporte Proteico/fisiologia , Raiva/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Animais , Asparagina/química , Asparagina/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glicosilação , Humanos , Camundongos , Células Tumorais CultivadasRESUMO
During 2014, henipavirus infection caused severe illness among humans and horses in southern Philippines; fatality rates among humans were high. Horse-to-human and human-to-human transmission occurred. The most likely source of horse infection was fruit bats. Ongoing surveillance is needed for rapid diagnosis, risk factor investigation, control measure implementation, and further virus characterization.
Assuntos
Surtos de Doenças , Infecções por Henipavirus/epidemiologia , Henipavirus/classificação , Adolescente , Adulto , Doenças dos Animais/epidemiologia , Doenças dos Animais/virologia , Animais , Sequência de Bases , Criança , Pré-Escolar , Feminino , Henipavirus/genética , Infecções por Henipavirus/diagnóstico , Infecções por Henipavirus/história , História do Século XXI , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Tipagem Molecular , Filipinas/epidemiologia , Filogenia , Vigilância da População , Alinhamento de Sequência , Sorotipagem , Proteínas Virais/química , Proteínas Virais/genética , Adulto JovemRESUMO
BACKGROUND: In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009-2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs). FINDINGS: Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. DISCUSSION: Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Virais/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/epidemiologia , Pandemias , Anticorpos de Cadeia Única/imunologia , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Antígenos Virais/genética , Antígenos Virais/imunologia , Bacteriófagos/genética , Bacteriófagos/imunologia , Sítios de Ligação , Células Clonais , Ensaio de Imunoadsorção Enzimática , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/virologia , Japão/epidemiologia , Biblioteca de Peptídeos , Sistemas Automatizados de Assistência Junto ao Leito/organização & administração , Ligação Proteica , Anticorpos de Cadeia Única/metabolismo , SolubilidadeRESUMO
A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay.