RESUMO
Hand, foot and mouth disease (HFMD) is an emerging infection with pandemic potential. Knowledge of neutralizing antibody responses among its pathogens is essential to inform vaccine development and epidemiologic research. We used 120 paired-plasma samples collected at enrollment and >7 days after the onset of illness from HFMD patients infected with enterovirus A71 (EV-A71), coxsackievirus A (CVA) 6, CVA10, and CVA16 to study cross neutralization. For homotypic viruses, seropositivity increased from <60% at enrollment to 97%-100% at follow-up, corresponding to seroconversion rates of 57%-93%. Seroconversion for heterotypic viruses was recorded in only 3%-23% of patients. All plasma samples from patients infected with EV-A71 subgenogroup B5 could neutralize the emerging EV-A71 subgenogroup C4. Collectively, our results support previous reports about the potential benefit of EV-A71 vaccine but highlight the necessity of multivalent vaccines to control HFMD.
Assuntos
Anticorpos Neutralizantes/imunologia , Enterovirus/imunologia , Doença de Mão, Pé e Boca/epidemiologia , Criança , Pré-Escolar , Feminino , Doença de Mão, Pé e Boca/sangue , Doença de Mão, Pé e Boca/prevenção & controle , Doença de Mão, Pé e Boca/virologia , Humanos , Lactente , Recém-Nascido , Masculino , Vietnã/epidemiologia , Vacinas ViraisRESUMO
We investigated enterovirus A71-associated hand, foot and mouth disease in Vietnam and found that, after replacing subgenogroup C4 in 2013, B5 remained the leading cause of this disease. In contrast with previous observations, this switch did not result in an explosive outbreak, and B5 evolution was driven by negative selection.
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Enterovirus Humano A/genética , Doença de Mão, Pé e Boca/virologia , Doença de Mão, Pé e Boca/epidemiologia , Humanos , Vietnã/epidemiologiaRESUMO
BACKGROUND: Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response. METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients. RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed. CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.
Assuntos
Infecções por Enterovirus/diagnóstico , Enterovirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Ásia , Criança , Pré-Escolar , Enterovirus/classificação , Enterovirus/genética , Feminino , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase Multiplex/normas , Faringe/virologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reto/virologia , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e EspecificidadeRESUMO
Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.
Assuntos
Citocinas/sangue , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Humana/sangue , Influenza Humana/mortalidade , RNA Viral/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Lactente , Influenza Humana/virologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
BACKGROUND: To date, little is known about the initial spread and response to the 2009 pandemic of novel influenza A ("2009 H1N1") in tropical countries. Here, we analyse the early progression of the epidemic from 26 May 2009 until the establishment of community transmission in the second half of July 2009 in Ho Chi Minh City (HCMC), Vietnam. In addition, we present detailed systematic viral clearance data on 292 isolated and treated patients and the first three cases of selection of resistant virus during treatment in Vietnam. METHODS AND FINDINGS: Data sources included all available health reports from the Ministry of Health and relevant health authorities as well as clinical and laboratory data from the first confirmed cases isolated at the Hospital for Tropical Diseases in HCMC. Extensive reverse transcription (RT)-PCR diagnostics on serial samples, viral culture, neuraminidase-inhibition testing, and sequencing were performed on a subset of 2009 H1N1 confirmed cases. Virological (PCR status, shedding) and epidemiological (incidence, isolation, discharge) data were combined to reconstruct the initial outbreak and the establishment of community transmission. From 27 April to 24 July 2009, approximately 760,000 passengers who entered HCMC on international flights were screened at the airport by a body temperature scan and symptom questionnaire. Approximately 0.15% of incoming passengers were intercepted, 200 of whom tested positive for 2009 H1N1 by RT-PCR. An additional 121 out of 169 nontravelers tested positive after self-reporting or contact tracing. These 321 patients spent 79% of their PCR-positive days in isolation; 60% of PCR-positive days were spent treated and in isolation. Influenza-like illness was noted in 61% of patients and no patients experienced pneumonia or severe outcomes. Viral clearance times were similar among patient groups with differing time intervals from illness onset to treatment, with estimated median clearance times between 2.6 and 2.8 d post-treatment for illness-to-treatment intervals of 1-4 d, and 2.0 d (95% confidence interval 1.5-2.5) when treatment was started on the first day of illness. CONCLUSIONS: The patients described here represent a cross-section of infected individuals that were identified by temperature screening and symptom questionnaires at the airport, as well as mildly symptomatic to moderately ill patients who self-reported to hospitals. Data are observational and, although they are suggestive, it is not possible to be certain whether the containment efforts delayed community transmission in Vietnam. Viral clearance data assessed by RT-PCR showed a rapid therapeutic response to oseltamivir.
Assuntos
Surtos de Doenças , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/epidemiologia , Programas de Rastreamento , Aeronaves , Transmissão de Doença Infecciosa/prevenção & controle , Transmissão de Doença Infecciosa/estatística & dados numéricos , Humanos , Incidência , Influenza Humana/tratamento farmacológico , Influenza Humana/transmissão , Oseltamivir/uso terapêutico , Fatores de Tempo , Viagem , Vietnã/epidemiologiaRESUMO
BACKGROUND: The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants. RESULTS: We developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10-100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%. CONCLUSIONS: This assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.
Assuntos
Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Humana/diagnóstico , Sondas de Oligonucleotídeos/genética , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência Conservada , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/virologia , Sensibilidade e Especificidade , VietnãRESUMO
Hand, foot and mouth disease (HFMD) continues to challenge Asia with pandemic potential. In Vietnam, there have been two major outbreaks occurring during 2011-2012 (>200,000 hospitalizations and >200 deaths) and more recently in 2018 (>130,000 hospitalizations and 17 deaths). Given the high burden and the complex epidemic dynamics of HFMD, synthesizing its clinical and epidemiological data remains essential to inform the development of appropriate interventions and design public health measures. We report the results of a hospital-based study conducted during 2015-2018, covering the severe HFMD outbreak recently documented in Vietnam in 2018. The study was conducted at three major hospitals responsible for receiving HFMD patients from southern Vietnam with a population of over 40 million. A total of 19 enterovirus serotypes were detected in 1196 HFMD patients enrolled in the clinical study during 2015-2018, with enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), CV-A10 and CV-A16 being the major causes. Despite the emergence of coxsackieviruses, EV-A71 remains the leading cause of severe HFMD in Vietnam. EV-A71 was consistently detected at a higher frequency during the second half of the years. The emergence of EV-A71 subgenogroup C4 in late 2018 was preceded by its low activity during 2017-early 2018. Compared with EV-A71 subgenogroup B5, C4 was more likely to be associated with severe HFMD, representing the first report demonstrating the difference in clinical severity between subgenogroup C4 and B5, the two predominant EV-A71 subgenogroups causing HFMD worldwide. Our data have provided significant insights into important aspects of HFMD over four years (2015-2018) in Vietnam, and emphasize active surveillance for pathogen circulation remains essential to inform the local public health authorities in the development of appropriate intervention strategies to reduce the burden of this emerging infections. Multivalent vaccines are urgently needed to control HFMD.
Assuntos
Doença de Mão, Pé e Boca/diagnóstico , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/etiologia , Criança , Pré-Escolar , Surtos de Doenças , Enterovirus/isolamento & purificação , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/etiologia , Infecções por Enterovirus/virologia , Feminino , Doença de Mão, Pé e Boca/virologia , Humanos , Lactente , Masculino , Sorogrupo , Vietnã/epidemiologiaRESUMO
BACKGROUND: New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. METHODS AND FINDINGS: Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1). CONCLUSIONS: These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/terapia , Adulto , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Feminino , Genoma Viral , Humanos , Imunização Passiva , Memória Imunológica/imunologia , Virus da Influenza A Subtipo H5N1/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , VietnãRESUMO
Japanese encephalitis virus was detected by deep sequencing for the first time in urine of a 16-year-old boy with encephalitis. Seroconversion and polymerase chain reaction analysis confirmed the metagenomics finding. Urine is useful for diagnosis of flaviviral encephalitis, whereas deep sequencing can be a panpathogen assay for the diagnosis of life-threatening infectious diseases.
RESUMO
BACKGROUND: Since 1962, enterovirus D68 (EV-D68) has been implicated in multiple outbreaks and sporadic cases of respiratory infection worldwide, but especially in the USA and Europe with an increasing frequency between 2010 and 2014. We describe the detection, associated clinical features and molecular characterization of EV-D68 in central and southern Viet Nam between 2009 and 2015. METHODS: Enterovirus/rhinovirus PCR positive respiratory or CSF samples taken from children and adults with respiratory/central nervous system infections in Viet Nam were tested by an EV-D68 specific PCR. The included samples were derived from 3 different observational studies conducted at referral hospitals across central and southern Viet Nam between 2009 and 2015. Whole-genome sequencing was carried out using a MiSeq based approach. Phylogenetic reconstruction and estimation of evolutionary rate and recombination were carried out in BEAST and Recombination Detection Program, respectively. RESULTS: EV-D68 was detected in 21/625 (3.4%) enterovirus/rhinovirus PCR positive respiratory samples but in none of the 15 CSF. All the EV-D68 patients were young children (age range: 11.8 - 24.5 months) and had moderate respiratory infections. Phylogenetic analysis suggested that the Vietnamese sequences clustered with those from Asian countries, of which 9 fell in the B1 clade, and the remaining sequence was identified within the A2 clade. One intra sub-clade recombination event was detected, representing the second reported recombination within EV-D68. The evolutionary rate of EV-D68 was estimated to be 5.12E -3 substitutions/site/year. Phylogenetic analysis indicated that the virus was imported into Viet Nam in 2008. CONCLUSIONS: We have demonstrated for the first time EV-D68 has been circulating at low levels in Viet Nam since 2008, associated with moderate acute respiratory infection in children. EV-D68 in Viet Nam is most closely related to Asian viruses, and clusters separately from recent US and European viruses that were suggested to be associated with acute flaccid paralysis.
RESUMO
Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.
Assuntos
Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Genoma Viral , Doença de Mão, Pé e Boca/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pré-Escolar , Enterovirus Humano A/isolamento & purificação , Variação Genética , Humanos , VietnãRESUMO
As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Mutação de Sentido Incorreto , Ligação Viral , Animais , Cristalografia por Raios X , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Aves Domésticas , Ligação Proteica , Conformação Proteica , RNA Viral/genética , Receptores Virais/metabolismo , Análise de Sequência de DNARESUMO
BACKGROUND: Since 2005, eight patients with H5N1 infection were laboratory confirmed in Cambodia. Despite the widespread of highly pathogenic avian influenza H5N1 virus and the intense exposure to poultry, there is growing evidence that H5N1 viruses may not be easily transmitted to human. OBJECTIVES: To evaluate the frequency of H5N1 transmission in rural Cambodia, to identify potential risk factors for H5N1 in humans and to explore the extent of asymptomatic and clinically mild illness among humans. STUDY DESIGN: A seroepidemiologic survey was conducted, 9 weeks after the recognition that H5N1 infection caused the death of a 13 years old female in April 2007. Blood specimens were collected from 700 participants for H5N1 serological testing. All participants were interviewed with standardized questionnaire to collect information about poultry exposure. RESULTS: Eighteen (2.6%) of the 700 villagers were tested positive cases for H5N1 antibodies. These 18 individuals were more likely than seronegative participants to report bathing or swimming in the community pond (p=0.04). CONCLUSIONS: The seroprevalence of H5N1 antibodies was higher than previously reported in the other investigations conducted in Cambodia and Thailand. This finding reinforces the overwhelming evidence that the virus continues to circulate widely in settings where human have high exposure to poultry. Our results, provides additional evidence suggesting that bathing or swimming in the community ponds, remains important potential risk factor for H5N1 infection. Both wild birds and domestic poultry have free access to these ponds which are also used for aquaculture through the dumping of poultry feces for fish feeding.
Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Influenza Humana/epidemiologia , Adolescente , Adulto , Idoso , Animais , Camboja/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , População Rural , Estudos Soroepidemiológicos , Inquéritos e Questionários , Adulto Jovem , Zoonoses/epidemiologia , Zoonoses/transmissãoRESUMO
BACKGROUND: Little is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection. METHODS: Using H5N1 microneutralisation (MN) and H5-pseudotype particle-based microneutralisation assays (H5pp) we analyzed sera sequentially obtained from 11 severely ill patients diagnosed by RT-PCR (follow-up range 1-139 weeks of disease onset) and 31 asymptomatically infected individuals detected in a sero-epidemiological study after exposure to H5N1 virus (follow-up range: 1-2 month-11 months after exposure). RESULTS: Of 44 sera from 11 patients with H5N1 disease, 70% tested positive by MN (antibody titre > or = 80) after 2 weeks and 100% were positive by 3 weeks after disease onset. The geometric mean MN titers in severely ill patients were 540 at 1-2 months and 173 at 10-12 months and thus were higher than the titers from asymptomatic individuals (149 at 1-2 months, 62.2 at 10-12 months). Fractional polynomial regression analysis demonstrated that in all severely ill patients, positive titers persisted beyond 2 years of disease onset, while 10 of 23 sera collected 10-11 months after exposure in asymptomatically infected individuals tested negative. CONCLUSIONS: Our results indicate that people with asymptomatic H5N1 infection have lower H5N1 antibody titres compared to those with severe illness and that in many asymptomatically infected patients the antibody titer decreased to levels below the threshold of positivity within one year. These data are essential for the design and interpretation of sero-epidemiological studies.
Assuntos
Anticorpos Neutralizantes/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/imunologia , Anticorpos Antivirais/imunologia , Camboja , Humanos , Virus da Influenza A Subtipo H5N1/classificação , Influenza Humana/sangue , Cinética , Testes de Neutralização , Análise de Regressão , Fatores de Tempo , VietnãRESUMO
Me Tri virus (MTV) is a member of the Semliki Forest virus (SFV) complex in the genus Alphavirus, first isolated from Culex tritaeniorhynchus mosquitoes in Vietnam in 1971 and described as a newly recognized alphavirus, based on antigenic characterization. However, based on a partial nucleotide sequence of the E1 envelope glycoprotein gene, it has recently been argued that MTV may represent a variant of SFV rather than a separate species. To enable definitive classification, we determined the complete genome sequence of MTV from original virus stock. Nucleotide homology, as well as phylogenetic analyses based on whole and partial genome sequences confirmed that MTV is an isolate of SFV. Notable differences to other reported SFV sequences included a 122 nt insertion at the 5' non-translated region (NTR), likely resulting from homologous recombination of part of the nsP2 gene, and differences in the sequence length of the 3' NTR. To our knowledge, this is the first and only documentation of SFV isolation outside Africa. Further research is needed to clarify whether SFV continues to circulate in Vietnam.
Assuntos
Vírus da Floresta de Semliki/classificação , Vírus da Floresta de Semliki/genética , Alphavirus/classificação , Alphavirus/genética , Animais , Sequência de Bases , Culex/virologia , DNA Viral/genética , Variação Genética , Dados de Sequência Molecular , Filogenia , Vírus da Floresta de Semliki/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , VietnãRESUMO
BACKGROUND: The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2. RESULTS: Using our assay, sera from patients who had recovered from infection with influenza H5N1, and sera from animals experimentally immunized or infected with H5 tested positive for the presence of neutralizing antibodies to H5N1. Pseudotype neutralizing antibody titers were compared with titers obtained by hemagglutinin inhibition (HI) assays and microneutralization (MN) assays using live virus, and showed a high degree of correlation, sensitivity and specificity. CONCLUSIONS: The pseudotype neutralization assay is as sensitive as horse erythrocyte HI and MN for the detection of antibodies to H5N1. It is safer, and can be applied in a high-throughput format for human and animal surveillance and for the evaluation of vaccines.