RESUMO
Echovirus 30 (E30) is an important causative agent of aseptic meningitis worldwide. Despite this, the global and regional dispersion patterns, especially in South America, are still largely unknown. We performed an in-depth analysis of global E30 population dynamics, by using the VP1 sequences of 79 strains isolated in Argentina, between 1998 and 2012, and 856 sequences from GenBank. Furthermore, the 3Dpol regions of 329 sequences were analyzed to study potential recombination events. E30 evolution was characterized by co-circulation and continuous replacement of lineages over time, where four lineages appear to circulate at present and another four lineages appear to have stopped circulating. Five lineages showed a global distribution, whereas three other lineages had a more restricted circulation pattern. Strains isolated in South America belong to lineages E and F. Analysis of the 3Dpol region of Argentinean strains indicated that recombination events occurred in both lineages.
Assuntos
Enterovirus Humano B/isolamento & purificação , Meningite Asséptica/virologia , Filogenia , América/epidemiologia , Ásia/epidemiologia , Enterovirus Humano B/classificação , Enterovirus Humano B/genética , Europa (Continente)/epidemiologia , Genótipo , Geografia , Humanos , Meningite Asséptica/epidemiologiaRESUMO
Human enteroviruses frequently cause severe diseases in children. Human enteroviruses are transmitted via the fecal-oral route and respiratory droplets, and primary replication occurs in the gastro-intestinal and respiratory tracts; however, how enteroviruses infect these sites is largely unknown. Human intestinal organoids have recently proven to be valuable tools for studying enterovirus-host interactions in the intestinal tract. In this study, we demonstrated the susceptibility of a newly developed human airway organoid model for enterovirus 71 (EV71) infection. We showed for the first time in a human physiological model that EV71 replication kinetics are strain-dependent. A glutamine at position 145 of the VP1 capsid protein was identified as a key determinant of infectivity, and residues VP1-98K and VP1-104D were identified as potential infectivity markers. The results from this study provide new insights into EV71 infectivity in the human airway epithelia and demonstrate the value of organoid technology for virus research.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/metabolismo , Infecções por Enterovirus/virologia , Organoides/virologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Enterovirus Humano A/química , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Humanos , Cinética , Alinhamento de Sequência , Virulência , Replicação ViralRESUMO
Enteroviruses (EVs) are among the most commonly detected viruses infecting humans worldwide. Although the prevalence of EVs is widely studied, the status of EV prevalence in sub-Saharan Africa remains largely unknown. The objective of our present study was therefore to increase our knowledge on EV circulation in sub-Saharan Africa. We obtained 749 fecal samples from a cross-sectional study conducted on Malawian children aged 6 to 60 months. We tested the samples for the presence of EVs using real time PCR, and typed the positive samples based on partial viral protein 1 (VP1) sequences. A large proportion of the samples was EV positive (89.9%). 12.9% of the typed samples belonged to EV species A (EV-A), 48.6% to species B (EV-B) and 38.5% to species C (EV-C). More than half of the EV-C strains (53%) belonged to subgroup C containing, among others, Poliovirus (PV) 1-3. The serotype most frequently isolated in our study was CVA-13, followed by EV-C99. The strains of CVA-13 showed a vast genetic diversity, possibly representing a new cluster, 'F'. The majority of the EV-C99 strains grouped together as cluster B. In conclusion, this study showed a vast circulation of EVs among Malawian children, with an EV prevalence of 89.9%. Identification of prevalences for species EV-C comparable to our study (38.5%) have only previously been reported in sub-Saharan Africa, and EV-C is rarely found outside of this region. The data found in this study are an important contribution to our current knowledge of EV epidemiology within sub-Saharan Africa.
Assuntos
Enterovirus Humano C/isolamento & purificação , Infecções por Enterovirus/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Criança , Pré-Escolar , Estudos de Coortes , Estudos Transversais , Enterovirus Humano C/classificação , Enterovirus Humano C/genética , Infecções por Enterovirus/epidemiologia , Fezes/virologia , Feminino , Variação Genética , Genótipo , Humanos , Lactente , Malaui/epidemiologia , Masculino , FilogeniaRESUMO
Human parechovirus 3 (HPeV3), a member of the Picornavirus family, is frequently detected worldwide. However, the observed seropositivity rates for HPeV3 neutralizing antibodies (nAbs) vary from high in Japan to low in the Netherlands and Finland. To study if this can be explained by technical differences or antigenic diversity among HPeV3 strains included in the serological studies, we determined the neutralizing activity of Japanese and Dutch intravenous immunoglobulin batches (IVIG), a rabbit HPeV3 hyperimmune polyclonal serum, and a human HPeV3-specific monoclonal antibody (mAb) AT12-015, against the HPeV3 A308/99 prototype strain and clinical isolates from Japan, the Netherlands and Australia, collected between 1989 and 2015. The rabbit antiserum neutralized all HPeV3 isolates whereas the neutralization capacity of the IVIG batches varied, and the mAb exclusively neutralized the A308/99 strain. Mapping of the amino acid variation among a subset of the HPeV3 strains on an HPeV3 capsid structure revealed that the majority of the surface-exposed amino acid variation was located in the VP1. Furthermore, amino acid mutations in a mAb AT12-015-resistant HPeV3 A308/99 variant indicated the location for potential antigenic determinants. Virus aggregation and the observed antigenic diversity in HPeV3 can explain the varying levels of nAb seropositivity reported in previous studies.
Assuntos
Anticorpos Neutralizantes/imunologia , Variação Antigênica/imunologia , Proteínas do Capsídeo/imunologia , Parechovirus/imunologia , Infecções por Picornaviridae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/genética , Variação Antigênica/genética , Proteínas do Capsídeo/genética , Humanos , Soros Imunes/imunologia , Japão , Mutação , Países Baixos , Testes de Neutralização , Parechovirus/classificação , Parechovirus/fisiologia , Infecções por Picornaviridae/virologia , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Outbreaks of human enterovirus 71 (EV-71) in Asia are related to high illness and death rates among children. To gain insight into the potential threat for the population of Europe, we determined the neutralizing activity in intravenous immunoglobulin (IVIg) batches and individual serum samples from donors in the Netherlands against EV-71 strains isolated in Europe and in Asia. All IVIg batches and 41%, 79%, and 65% of serum samples from children ≤5 years of age, women of childbearing age, and HIV-positive men, respectively, showed high neutralizing activity against a Dutch C1 strain, confirming widespread circulation of EV-71 in the Netherlands. Asian B3-4 and C4 strains were efficiently cross-neutralized, predicting possible protection against extensive circulation and associated outbreaks of those types in Europe. However, C2 and C5 strains that had few mutations in the capsid region consistently escaped neutralization, emphasizing the importance of monitoring antigenic diversity among circulating EV-71 strains.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/imunologia , Adulto , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Linhagem Celular , Pré-Escolar , Coinfecção , Proteção Cruzada/imunologia , Surtos de Doenças , Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Infecções por Enterovirus/prevenção & controle , Infecções por Enterovirus/virologia , Feminino , Genótipo , Infecções por HIV , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Testes de Neutralização , Vigilância da População , Estudos Soroepidemiológicos , Ensaio de Placa Viral , Adulto JovemRESUMO
UNLABELLED: Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCE: Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.
Assuntos
Poliomielite/prevenção & controle , Vacinas contra Poliovirus/isolamento & purificação , Poliovirus/isolamento & purificação , Poliovirus/fisiologia , Tecnologia Farmacêutica/métodos , Replicação Viral , Animais , Vacinas Atenuadas/isolamento & purificação , Células Vero , Cultura de Vírus/métodosRESUMO
The incidence of coxsackievirus A6 (CV-A6) associated hand, foot and mouth disease (HFMD) has reportedly increased since 2008 with sometimes severe complications. We here describe an atypical course of CV-A6-associated HFMD in extremely low birth weight twins. The CV-A6-strains are genetically closely related to international strains isolated from HFMD outbreaks.
Assuntos
Doenças em Gêmeos/diagnóstico , Enterovirus/isolamento & purificação , Doença de Mão, Pé e Boca/diagnóstico , Recém-Nascido de Peso Extremamente Baixo ao Nascer , Recém-Nascido de Baixo Peso , Doenças do Prematuro/diagnóstico , Doenças em Gêmeos/virologia , Enterovirus/classificação , Feminino , Doença de Mão, Pé e Boca/virologia , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/virologia , Masculino , Filogenia , Fatores de Risco , GêmeosRESUMO
BACKGROUND: Since 1997, several countries within the Asian Pacific region have been affected by one or more massive outbreaks of Hand Foot and Mouth Disease (HFMD). Virus typing experiments revealed that these outbreaks were caused by strains of human enterovirus 71 (EV71) belonging to several different, recently emerged subgenogroups. In mainland China, a different situation was observed. The first outbreak, localized in Shangdong Province, was reported in 2007, and was followed by a wide-spread outbreak in mainland China in 2008. Since then, numbers of reported HFMD cases have been persistently high. METHODOLOGY/PRINCIPAL FINDINGS: To gain insight in the epidemiological behavior of EV71 in China, we studied genetic diversity and EV71 population dynamics to address whether the increase in number of reported EV71 infections reflects a real increase in viral spread or is just the result of increased awareness and surveillance. We used systematically collected VP1 gene sequences of 257 EV71 strains collected in Guangdong province from 2008 to 2010 as part of HFMD surveillance activities, and supplemented them with 305 GenBank EV71 reference stains collected in China from 1998 to 2010. All isolates from Guangdong Province belonged to subgenogroup C4. Viral population dynamics indicated that the increased reporting of HFMD in China since 2007 reflects a real increase in viral spread and continued replacement of viral lineages through time. Amino acid sequence comparisons revealed substitution of amino acid in residues 22, 145 and 289 through time regularly with the VP1 gene of EV71 strains isolated in mainland China from 1998 to 2010. CONCLUSIONS: EV71 strains isolated in mainland China mainly belonged to subgenogroup C4. There was exponential growth of the EV71 virus population in 2007 and 2008. There was amino acid substitution through time regularly with the VP1 gene which possibly increased viral spread and/or ability of the virus to circulate persistently among the Chinese population.
Assuntos
Enterovirus Humano A/genética , Variação Genética , Algoritmos , Sequência de Aminoácidos , Teorema de Bayes , China/epidemiologia , DNA Viral , Surtos de Doenças , Enterovirus Humano A/isolamento & purificação , Doença de Mão, Pé e Boca/virologia , Humanos , Dados de Sequência Molecular , Método de Monte Carlo , Mutação , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Following an increase in detection of enterovirus 68 (EV68) in community surveillance of respiratory infections in The Netherlands in 2010, epidemiological and virological analyses were performed to investigate the possible public health impact of EV68 infections. We retrospectively tested specimens collected from acute respiratory infections surveillance and through three children cohort studies conducted in The Netherlands from 1994 through 2010. A total of 71 of 13,310 (0.5%) specimens were positive for EV68, of which 67 (94%) were from symptomatic persons. Twenty-four (34%) of the EV68 positive specimens were collected during 2010. EV68-positive patients with respiratory symptoms showed significantly more dyspnea, cough and bronchitis than EV68-negative patients with respiratory symptoms. Phylogenetic analysis showed an increased VP1 gene diversity in 2010, suggesting that the increased number of EV68 detections in 2010 reflects a real epidemic. Clinical laboratories should consider enterovirus diagnostics in the differential diagnosis of patients presenting with respiratory symptoms.
Assuntos
Enterovirus Humano D/isolamento & purificação , Infecções por Enterovirus/epidemiologia , Infecções Respiratórias/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Enterovirus Humano D/classificação , Enterovirus Humano D/genética , Infecções por Enterovirus/virologia , Epidemias , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Países Baixos/epidemiologia , Filogenia , Infecções Respiratórias/virologia , Estudos Retrospectivos , Adulto JovemRESUMO
Evolutionary history of human enterovirus 71 (EV71) in the Netherlands shows displacement of virus subgenogroups, that only partly can be explained by antigenic changes. Additionally, occasional epidemics have occurred that remain to be explained. Previous studies have shown subgenogroup specific recombination events in the genome of Asian EV71 strains. To find clues on the role of genome recombination in evolution of the EV71 subgenogroups found in Europe and in the evolution of strains capable of causing outbreaks, we analyzed the genomes of 19 strains representing the genetic diversity of EV71 in the Netherlands between 1963 and 2010. We selected viruses from EV71 endemic and epidemic years (1986 and 2007). Subgenogroup specific genome recombination events were detected for subgenogroup B0, B1 and B2 viruses, in line with observed genome recombination events in Asian subgenogroup B3 and B4 viruses. Considering recombination events distinguishing strains from epidemic years from those of non-epidemic years, breakpoints for recombination were detected in the 5'UTR of B2 viruses from the outbreak in 1986, with highest similarity of the 5'UTR to B4 and B3 strains isolated during outbreaks in the Asian Pacific region. No indications for recombination were found in genogroup C isolates. Except for the '86 B2 isolates' Dutch isolates phylogenetically interspersed with international reference strains of the same subgenogroup, indicating a global dissemination of (recombinant) EV71 viruses. The difference observed in the 5'UTR of EV71 strains isolated in endemic versus epidemic years suggests that changes in the 5'UTR cause evolution of strains capable of causing outbreaks.
Assuntos
Enterovirus Humano A/genética , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Enterovirus Humano A/isolamento & purificação , Epidemias , Genoma , Genoma Viral , Humanos , Países Baixos/epidemiologia , Filogenia , Vírus ReordenadosRESUMO
From 1963 to 1986, human enterovirus 71 (HEV71) infections in the Netherlands were successively caused by viruses of subgenogroups B0, B1 and B2. A genogroup shift occurred in 1987, after which viruses of subgenogroups C1 and C2 were detected exclusively. This is in line with HEV71 typing in Australia, Europe and the USA, but is distinct from that in the Asian Pacific region, where HEV71 subgenogroups B3-B5 and C4-C5 have caused large outbreaks since 1997. To understand these observations in HEV71 epidemiology, the VP1-encoding regions of 199 HEV71 strains isolated in the Netherlands between 1963 and 2008 were used to study the detailed evolutionary trajectory and population dynamics of HEV71. Genogroup B viruses showed an epochal evolution, whereas genogroup C viruses evolved independently, which is in line with the co-circulation of C1 and C2 viruses in the Netherlands since 1997. Considering that strains from the Netherlands are interspersed phylogenetically with GenBank reference strains, the evolution of B1-B2, C1-C2 viruses has a global nature. Phylodynamic analysis confirmed that increased reporting of HEV71 infections in 1986 and 2007 reflected true epidemics of B2 and C2 viruses, respectively. Sequence analysis of the complete capsid region of a subset of isolates revealed several (sub)genogroup-specific residues. Subgenogroup B2-specific rabbit antiserum showed cross-neutralization of B0, B1 and B2 viruses, but not of subgenogroup C1 or C2 viruses, probably explaining the global shift to genogroup C in 1987 following a B2 epidemic. Anti-C1 rabbit serum neutralized both genogroup B and C viruses. Global herd immunity against C1 and C2 viruses possibly explains why epidemics with subgenogroups B4 and C4 are restricted to the Asian Pacific region.
Assuntos
Proteínas do Capsídeo/genética , Enterovirus Humano A/genética , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Evolução Molecular , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Reações Cruzadas , Enterovirus Humano A/classificação , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Genótipo , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Países Baixos/epidemiologia , RNA Viral/genética , Coelhos , Análise de Sequência de DNARESUMO
The incidence of enterovirus 71 (EV71) infection has greatly increased in the Asian Pacific region since 1997. Several large outbreaks, caused by different subgenogroups of EV71, occurred with high rates of morbidity and a substantial number of deaths. In 2007, 58 cases of EV71 infection requiring hospitalization were reported in The Netherlands after a period of low endemicity of 21 years. These events triggered a study on the epidemiology of EV71 in The Netherlands. Genetic analysis of the VP1 capsid region of 199 EV71 isolates collected from 1963 to 2008 as part of enterovirus surveillance activities revealed a change in the prevailing subgenogroups over time. From 1963 to 1986 infections were caused by three different and successive lineages belonging to subgenogroup B (the novel lineage designated B0, as well as B1 and B2). In 1987, following a major epidemic the previous year, the B genogroup was replaced by genogroup C strains of lineages C1 and, later, C2. Analyses of the clinical data suggested that there were differences between infection with genogroup B and with genogroup C strains in terms of the age groups affected and the severity of illness. From comparative analysis with genomic data available in the public domain, we concluded that EV71 strain evolution shows a global pattern, which leads to the question of whether the recently emerged C4 lineage strains will also spread outside of Asia.
Assuntos
Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Animais , Proteínas do Capsídeo/genética , Criança , Pré-Escolar , Análise por Conglomerados , Enterovirus Humano A/genética , Genótipo , Humanos , Incidência , Dados de Sequência Molecular , Países Baixos/epidemiologia , Filogenia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
For the final stages in the eradication of poliovirus type 1 (P1), the World Health Organization advocates the selective use of monovalent type 1 oral poliovirus vaccine (mOPV1). To compare the immunogenicity of mOPV1 with that of trivalent OPV (tOPV) in infants, a study was performed in Egypt in 2005. Newborns were vaccinated with mOPV1 or tOPV immediately after birth and were challenged with mOPV1 after 1 month. Vaccination with mOPV1 at birth resulted in significantly higher seroconversion against P1 viruses and lower excretion of P1 viruses than vaccination with tOPV. Intratypic differentiation of the viruses shed by the newborns revealed the presence of remarkably high numbers of antigenically divergent (AD) P1 isolates, especially in the mOPV1 study group. The majority of these AD P1 isolates (71%) were mOPV1 challenge derived and were shed by newborns who did not seroconvert to P1 after the birth dose. Genetic characterization of the viruses revealed that amino acid 60 of the VP3 region was mutated in all AD P1 isolates. Isolates with substitution of residue 99 of the VP1 region had significantly higher numbers of nonsynonymous mutations in the VP1 region than isolates without this substitution and were preferentially shed in the mOPV1 study group. The widespread use of mOPV1 has proven to be a powerful tool for fighting poliovirus circulation in the remaining areas of endemicity. This study provides another justification for the need to achieve high vaccination coverage in order to prevent the circulation of AD strains.
Assuntos
Antígenos Virais/genética , Antígenos Virais/imunologia , Variação Genética , Vacina Antipólio Oral/administração & dosagem , Poliovirus/genética , Poliovirus/imunologia , Eliminação de Partículas Virais , Substituição de Aminoácidos/genética , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Egito , Humanos , Lactente , Recém-Nascido , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Poliovirus/classificação , Poliovirus/isolamento & purificação , Análise de Sequência de DNARESUMO
Infection with human parechovirus 3 (HPeV3) was described for the first time in Japan in 2004 and reportedly is more often associated with severe disease than infection with HPeV1 or HPeV2. In 2004, infections with HPeV3 were observed for the first time in The Netherlands. Genetic analysis showed several different lineages, suggesting endemic circulation. We analyzed 163 cell culture isolates from the same number of patients tested in routine virological laboratories as part of the national enterovirus surveillance program. Isolates were collected between 2000 and 2007 and could not be characterized by routine methods. In total, 155 isolates (95%) were found positive for HPeV by a reverse transcription-PCR assay targeting the 5' untranslated region, explaining the majority of the diagnostic deficit in enterovirus surveillance for these years. Typing of the isolates by use of partial genome sequencing of the VP1/2A region revealed the presence of 55 HPeV1, 2 HPeV2, 89 HPeV3, 1 HPeV4, and 8 HPeV5 isolates. We compared isolation dates, age groups affected, and clinical pictures, which were reported as part of the routine surveillance. Clear differences in epidemiology were observed, with HPeV3 occurring at intervals of 2 years and in the spring-summer season, whereas HPeV1 was observed in small numbers throughout each year, with a low in the summer months. HPeV3 infection affected younger children than HPeV1 infection and was significantly more often associated with fever, meningitis, and viremia.