RESUMO
This report aims to evaluate the usefulness of self-sampling as an approach for future national surveillance of emerging respiratory infections by comparing virological data from two parallel surveillance schemes in England. Nasal swabs were obtained via self-administered sampling from consenting adults (≥ 16 years-old) with influenza symptoms who had contacted the National Pandemic Flu Service (NPFS) health line during the 2009 influenza pandemic. Equivalent samples submitted by sentinel general practitioners participating in the national influenza surveillance scheme run jointly by the Royal College of General Practitioners (RCGP) and Health Protection Agency were also obtained. When comparable samples were analysed there was no significant difference in results obtained from self-sampling and clinician-led sampling schemes. These results demonstrate that self-sampling can be applied in a responsive and flexible manner, to supplement sentinel clinician-based sampling, to achieve a wide spread and geographically representative way of assessing community transmission of a known organism.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , Autoadministração/métodos , Vigilância de Evento Sentinela , Manejo de Espécimes/métodos , Adulto , Inglaterra/epidemiologia , Feminino , Humanos , Influenza Humana/diagnóstico , Influenza Humana/epidemiologia , Masculino , Cavidade Nasal/virologia , Pandemias , RNA Viral/genética , Características de Residência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inquéritos e QuestionáriosRESUMO
During the 2009 influenza A(H1N1) pandemic, a new laboratory-based virological sentinel surveillance system, the Respiratory DataMart System (RDMS), was established in a network of 14 Health Protection Agency (now Public Health England (PHE)) and National Health Service (NHS) laboratories in England. Laboratory results (both positive and negative) were systematically collected from all routinely tested clinical respiratory samples for a range of respiratory viruses including influenza, respiratory syncytial virus (RSV), rhinovirus, parainfluenza virus, adenovirus and human metapneumovirus (hMPV). The RDMS also monitored the occurrence of antiviral resistance of influenza viruses. Data from the RDMS for the 20092012 period showed that the 2009 pandemic influenza virus caused three waves of activity with different intensities during the pandemic and post pandemic periods. Peaks in influenza A(H1N1)pdm09 positivity (defined as number of positive samples per total number of samples tested) were seen in summer and autumn in 2009, with slightly higher peak positivity observed in the first post-pandemic season in 2010/2011. The influenza A(H1N1)pdm09 virus strain almost completely disappeared in the second postpandemic season in 2011/2012. The RDMS findings are consistent with other existing community-based virological and clinical surveillance systems. With a large sample size, this new system provides a robust supplementary mechanism, through the collection of routinely available laboratory data at minimum extra cost, to monitor influenza as well as other respiratory virus activity. A near real-time, daily reporting mechanism in the RDMS was established during the London 2012 Olympic and Paralympic Games. Furthermore, this system can be quickly adapted and used to monitor future influenza pandemics and other major outbreaks of respiratory infectious disease, including novel pathogens.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Laboratórios/estatística & dados numéricos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/isolamento & purificação , Vigilância de Evento Sentinela , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Inglaterra/epidemiologia , Feminino , Humanos , Lactente , Influenza Humana/epidemiologia , Influenza Humana/virologia , Masculino , Notificação de Abuso , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Estações do Ano , Adulto JovemRESUMO
REASONS FOR PERFORMING STUDY: The use of tongue ties in racing is common, yet there are few data to support their efficacy. In order to make appropriate recommendations for clinical practice and policy on tongue ties, data documenting their effect on upper airway structure are necessary. OBJECTIVES: To determine the effect of a tongue tie on the resting laryngohyoid position of the standing horse. STUDY DESIGN: Experimental study. METHODS: Twelve normal Standardbred horses were subjected to ultrasonographic measures of laryngohyoid position during 3 phases of tack application: Phase I--halter and lead; Phase II--bit, bridle, harness and check applied; and Phase III--as in Phase II but with the tongue tie added. RESULTS: Compared to Phase I, during Phase III with the application of a tongue tie a significant difference between lingual process position was noted both rostrally and caudally (P<0.001 and P<0.001), such that the tongue tie resulted in an increase in lingual process depth. The tongue tie in Phase III resulted in a decrease in depth of the thyroid cartilage and basihyoid bone compared to the Phase I location (P = 0.007 and P = 0.0047). CONCLUSION: The use of a tongue tie has a significant effect on the basihyoid and thyroid cartilage positions in the standing horse. POTENTIAL RELEVANCE: This is the first report documenting a difference in laryngohyoid morphology following the application of a tongue tie, providing evidence that the use of a tongue tie has a measurable effect on upper airway structure. The functional implications of this finding are yet to be elucidated.
Assuntos
Cavalos , Osso Hioide/anatomia & histologia , Laringe/anatomia & histologia , Língua , Animais , Feminino , MasculinoRESUMO
On 22 September 2012, a novel coronavirus, very closely related to that from a fatal case in Saudi Arabia three months previously, was detected in a previously well adult transferred to intensive care in London from Qatar with severe respiratory illness. Strict respiratory isolation was instituted. Ten days after last exposure, none of 64 close contacts had developed severe disease, with 13 of 64 reporting mild respiratory symptoms. The novel coronavirus was not detected in 10 of 10 symptomatic contacts tested.
Assuntos
Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Coronavirus/isolamento & purificação , Prática de Saúde Pública , Síndrome Respiratória Aguda Grave/diagnóstico , Viagem , Adulto , Infecções por Coronavirus/virologia , Humanos , Londres , Masculino , Arábia Saudita , Síndrome Respiratória Aguda Grave/virologia , Reino UnidoRESUMO
Coronaviruses have the potential to cause severe transmissible human disease, as demonstrated by the severe acute respiratory syndrome (SARS) outbreak of 2003. We describe here the clinical and virological features of a novel coronavirus infection causing severe respiratory illness in a patient transferred to London, United Kingdom, from the Gulf region of the Middle East.
Assuntos
Infecções por Coronavirus/diagnóstico , Coronavirus/isolamento & purificação , Transferência de Pacientes , Síndrome Respiratória Aguda Grave/etiologia , Viagem , Animais , Coronavirus/classificação , Coronavirus/patogenicidade , Infecções por Coronavirus/microbiologia , Infecções por Coronavirus/virologia , Notificação de Doenças , Reservatórios de Doenças , Perfilação da Expressão Gênica , Humanos , Unidades de Terapia Intensiva , Londres , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Insuficiência Respiratória/complicações , Insuficiência Respiratória/terapia , Arábia Saudita , Sensibilidade e Especificidade , Síndrome Respiratória Aguda Grave/microbiologia , Síndrome Respiratória Aguda Grave/terapiaRESUMO
We describe an outbreak of human metapneumovirus (hMPV) which occurred in July-September 2010 at a community hospital in the East of England. Based on the medical and nursing records, cases were retrospectively defined as suspected if they had had an influenza-like illness (ILI), and probable if they had had an ILI and an epidemiological link to a laboratory-confirmed case. Of a total of 17 symptomatic inpatients, five were classified as probable cases, five were laboratory confirmed and seven were suspected. The attack rate was 29.4% for confirmed and probable cases combined. The median age of symptomatic inpatients was 85 years-old (range 68-96) and the majority (16/17) of symptomatic inpatients had an underlying medical condition. Control measures introduced appeared to restrict further exposure of susceptible patients to infection although modelling suggested that up to four of 10 confirmed and probable cases (40%) could have been prevented through more timely diagnosis and recognition of an outbreak. These findings suggest that there should be increased awareness of hMPV infection within healthcare settings, particularly when the population at risk has a high prevalence of underlying co-morbidities.
Assuntos
Surtos de Doenças , Metapneumovirus/isolamento & purificação , Infecções por Paramyxoviridae/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/virologia , Inglaterra/epidemiologia , Feminino , Hospitais Comunitários , Humanos , Incidência , Masculino , Metapneumovirus/genética , Infecções por Paramyxoviridae/diagnóstico , Infecções por Paramyxoviridae/transmissão , Infecções por Paramyxoviridae/virologia , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Fatores de Risco , Fatores de TempoAssuntos
Macrolídeos/uso terapêutico , Mycoplasma pneumoniae/isolamento & purificação , Pneumonia por Mycoplasma/diagnóstico , Pneumonia por Mycoplasma/epidemiologia , Adolescente , Criança , Pré-Escolar , Inglaterra/epidemiologia , Feminino , Marcadores Genéticos , Humanos , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Mycoplasma pneumoniae/efeitos dos fármacos , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/tratamento farmacológico , Vigilância da População , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Sequências de Repetição em Tandem , País de Gales/epidemiologiaRESUMO
Real-time PCR was employed to detect a conserved region of the P1 cytadhesin gene of Mycoplasma pneumoniae in combined nose and throat swabs collected from patients attending GP surgeries during 2005-2009 with symptoms of respiratory tract infection (RTI). Samples were collected as part of an annual winter epidemiological and virological linked study in England and Wales. A total of 3,987 samples were tested, 65 (1.7%, 95%CI 1.3-2.1) had detectable M. pneumoniae DNA. Positive patients were detected of both gender, aged from 9 months to 78 years, who had clinical signs of upper RTI, fever and/or myalgia, an influenza-like illness to lower RTI. Mixed infections were identified in four cases, two with influenza A H1, one with H3 and one with influenza B. Children aged 5-14 years were more likely to have detectable M. pneumoniae in samples than all other age groups (Fishers p = 0.03), attributed to the 2005-2006 season in which 6.0% (12/200, 95%CI 3.4-10.3) of 5-14 year olds had detectable M. pneumoniae in comparison to 2.2% in 2006-2007 (3/141 95%CI 0.5-6.4), 2.2% in 2007-2008 (2/89 95%CI 0.1-8.3) and 0% in 2008-2009 (0/151 95%CI 0-2.9).
Assuntos
Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecções por Mycoplasma/epidemiologia , Mycoplasma pneumoniae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Infecções Respiratórias/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Inglaterra/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Infecções por Mycoplasma/microbiologia , Nariz/microbiologia , Faringe/microbiologia , Atenção Primária à Saúde , Infecções Respiratórias/microbiologia , País de Gales/epidemiologia , Adulto JovemRESUMO
During the winter period 2010/11 27 epidemiologically unlinked, confirmed cases of oseltamivir-resistant influenza A(H1N1)2009 virus infection have been detected in multiple, geographically dispersed settings. Three of these cases were in community settings, with no known exposure to oseltamivir. This suggests possible onward transmission of resistant strains and could be an indication of a possibility of changing epidemiology of oseltamivir-resistant influenza A(H1N1)2009 virus.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/epidemiologia , Oseltamivir/uso terapêutico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/tratamento farmacológico , Influenza Humana/transmissão , Influenza Humana/virologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Neuraminidase/genética , Pandemias , Polimorfismo de Nucleotídeo Único , Vigilância da População , Estações do Ano , Reino Unido/epidemiologia , Adulto JovemRESUMO
The 2010/11 winter influenza season is underway in the United Kingdom, with co-circulation of influenza A(H1N1)2009 (antigenically similar to the current 2010/11 vaccine strain), influenza B (mainly B/Victoria/2/87 lineage, similar to the 2010/11 vaccine strain) and a few sporadic influenza A(H3N2) viruses. Clinical influenza activity has been increasing. Severe illness, resulting in hospitalisation and deaths, has occurred in children and young adults and has predominantly been associated with influenza A(H1N1)2009, but also influenza B viruses.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza B/genética , Influenza Humana/mortalidade , Influenza Humana/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais/genética , Criança , Surtos de Doenças , Feminino , Genótipo , Hospitalização , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza B/imunologia , Vírus da Influenza B/isolamento & purificação , Vacinas contra Influenza/imunologia , Influenza Humana/diagnóstico , Influenza Humana/prevenção & controle , Masculino , Pessoa de Meia-Idade , Fenótipo , Filogenia , Estações do Ano , Vigilância de Evento Sentinela , Análise de Sequência de DNA , Índice de Gravidade de Doença , Reino Unido/epidemiologia , Adulto JovemRESUMO
Uncertainties exist regarding the population risks of hospitalization due to pandemic influenza A(H1N1). Understanding these risks is important for patients, clinicians and policy makers. This study aimed to clarify these uncertainties. A national surveillance system was established for patients hospitalized with laboratory-confirmed pandemic influenza A(H1N1) in England. Information was captured on demographics, pre-existing conditions, treatment and outcomes. The relative risks of hospitalization associated with pre-existing conditions were estimated by combining the captured data with population prevalence estimates. A total of 2416 hospitalizations were reported up to 6 January 2010. Within the population, 4·7 people/100,000 were hospitalized with pandemic influenza A(H1N1). The estimated hospitalization rate of cases showed a U-shaped distribution with age. Chronic kidney disease, chronic neurological disease, chronic respiratory disease and immunosuppression were each associated with a 10- to 20-fold increased risk of hospitalization. Patients who received antiviral medication within 48 h of symptom onset were less likely to be admitted to critical care than those who received them after this time (adjusted odds ratio 0·64, 95% confidence interval 0·44-0·94, P=0·024). In England the risk of hospitalization with pandemic influenza A(H1N1) has been concentrated in the young and those with pre-existing conditions. By quantifying these risks, this study will prove useful in planning for the next winter in the northern and southern hemispheres, and for future pandemics.
Assuntos
Hospitalização/estatística & dados numéricos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/epidemiologia , Pandemias , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Comorbidade , Inglaterra/epidemiologia , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Influenza Humana/patologia , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Adulto JovemRESUMO
This paper describes the epidemiology of fatal pandemic influenza A(H1N1) cases in the United Kingdom (UK) since April 2009 and in particular risk factors associated with death. A fatal case was defined as a UK resident who died between 27 April 2009 and 12 March 2010, in whom pandemic influenza A(H1N1) infection was confirmed by laboratory or recorded on death certificate. Case fatality ratios (CFR) were calculated using the estimated cumulative number of clinical cases as the denominator. The relative risk of death was estimated by comparing the population mortality rate in each risk group, with those not in a risk group. Across the UK, 440 fatal cases were identified. In England, fatal cases were mainly seen in young adults (median age 43 years, 85% under 65 years), unlike for seasonal influenza. The majority (77%) of cases for whom data were available (n=308) had underlying risk factors for severe disease. The CFR in those aged 65 years or over was nine per 1,000 (range 3 - 26) compared to 0.4 (range 0.2 to 0.9) for those aged six months to 64 years. In the age group between six month and 64 years, the relative risk for fatal illness for those in a risk group was 18. The population attributable fractions in this age group were highest for chronic neurological disease (24%), immunosuppression (16%) and respiratory disease (15%). The results highlight the importance of early targeted effective intervention programmes.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana/mortalidade , Mortalidade/tendências , Adolescente , Adulto , Idoso , Surtos de Doenças , Estudos Epidemiológicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Reino Unido/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Estimation of influenza vaccine effectiveness (V/E) is needed early during influenza outbreaks in order to optimise management of influenza--a need which will be even greater in a pandemic situation. OBJECTIVE: Examine the potential of routinely collected virological surveillance data to generate estimates of V/E in real-time during winter seasons. METHODS: Integrated clinical and virological community influenza surveillance data collected over three winters 2004/5-2006/7 were used. We calculated the odds of vaccination in persons that were influenza-virus-positive and the odds in those that were negative and provided a crude estimate of V/E. Logistic regression was used to obtain V/E estimates adjusted for confounding variables such as age. RESULTS: Multivariable analysis suggested that adjustments to the crude V/E estimate were necessary for patient age and month of sampling. The annual adjusted V/E was 2005/6, 67% (95% CI 41% to 82%); 2006/7 55% (26% to 73%) and 2007/8 67% (41% to 82%). The adjusted V/E in persons <65 years was 70% (57% to 78%) and 65 years and over 46% (-17% to 75%). Estimates differed by small insignificant amounts when calculated separately for influenza A and B; by interval between illness onset and swab sample; by analysis for the period November to January in each year compared with February to April and according to viral load. CONCLUSION: We have demonstrated the potential of using routine virological and clinical surveillance data to provide estimates of V/E early in season and conclude that it is feasible to introduce this approach to V/E measurement into evaluation of national influenza vaccination programs.
Assuntos
Vacinas contra Influenza , Influenza Humana/epidemiologia , Infecções Respiratórias/diagnóstico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Humanos , Incidência , Lactente , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/prevenção & controle , Pessoa de Meia-Idade , Análise Multivariada , Nasofaringe/virologia , Vigilância da População , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/virologia , Estações do Ano , Reino Unido/epidemiologia , Carga Viral/estatística & dados numéricosRESUMO
On 29 April 2009, an imported case of pandemic (H1N1) 2009 virus infection was detected in a London school. As further cases, pupils and staff members were identified, school closure and mass prophylaxis were implemented. An observational descriptive study was conducted to provide an insight into the clinical presentation and transmission dynamics in this setting. Between 15 April and 15 May 2009, 91 symptomatic cases were identified: 33 were confirmed positive for pandemic (H1N1) 2009 virus infection; 57 were tested negative; in one the results were unavailable. Transmission occurred first within the school, and subsequently outside. Attack rates were 2% in pupils (15% in the 11-12 years age group) and 17% in household contacts. The predominant symptoms were fever (97%), respiratory symptoms (91%), and sore throat (79%). Limited spread in the school may have been due to a combination of school closure and mass prophylaxis. However, transmission continued through household contacts to other schools.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana/epidemiologia , Adolescente , Antivirais/uso terapêutico , Criança , Surtos de Doenças , Feminino , Humanos , Influenza Humana/prevenção & controle , Influenza Humana/transmissão , Londres/epidemiologia , Masculino , Oseltamivir/uso terapêutico , Instituições Acadêmicas , Adulto JovemRESUMO
The sensitivity and specificity of four real-time PCR assays (HPA A(H1)v, CDC A (H1)v, HPA A(N1)v and NVRL S-OIV assays) was evaluated for detection of influenza A(H1N1)v viruses. Nose and throat swab samples containing influenza A(H1N1)v viruses, seasonal influenza AH3N2, AH1N1, influenza B viruses, or negative for influenza viruses were tested by the four assays. Specificity was also analysed using influenza A viruses of different subtypes and non-related respiratory viruses. The sensitivities and specificities of the four assays were in a similar range and suitable for diagnostic use. The HPA (H1)v and the S-OIV assays were the most sensitive assays for use as a first line test, but the S-OIV assay was less specific, detecting all avian subtypes of influenza A viruses tested. The results of this study demonstrate that the concurrent use of primary diagnostic and confirmatory assays provides rapid and accurate assessment of confirmed cases, and allows appropriate management of patients.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Vigilância da População/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The emergence of new viral infections of man requires the development of robust diagnostic tests that can be applied in the differential diagnosis of acute illness, or to determine past exposure, so as to establish the true burden of disease. Since the recognition in April 2003 of the severe acute respiratory syndrome coronavirus (SARS-CoV) as the causative agent of severe acute respiratory syndrome (SARS), enormous efforts have been applied to develop molecular and serological tests for SARS which can assist rapid detection of cases, accurate diagnosis of illness and the application of control measures. International progress in the laboratory diagnosis of SARS-CoV infection during acute illness has led to internationally agreed World Health Organization criteria for the confirmation of SARS. Developments in the dissection of the human immune response to SARS indicate that serological tests on convalescent sera are essential to confirm SARS infection, given the sub-optimal predictive value of molecular detection tests performed during acute SARS illness.
Assuntos
Anticorpos Antivirais/imunologia , Síndrome Respiratória Aguda Grave/diagnóstico , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Humanos , Imunoensaio/métodos , Técnicas de Sonda Molecular , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Testes Sorológicos/métodos , Síndrome Respiratória Aguda Grave/imunologiaRESUMO
Mutant recombinant respiratory syncytial viruses (RSV) which cannot express the NS1 and M2-2 proteins, designated rA2DeltaNS1 and rA2DeltaM2-2, respectively, were evaluated as live-attenuated RSV vaccines. The rA2DeltaNS1 virus contains a large deletion that should have the advantageous property of genetic stability during replication in vitro and in vivo. In vitro, rA2DeltaNS1 replicated approximately 10-fold less well than wild-type recombinant RSV (rA2), while rA2DeltaM2-2 had delayed growth kinetics but reached a final titer similar to that of rA2. Each virus was administered to the respiratory tracts of RSV-seronegative chimpanzees to assess replication, immunogenicity, and protective efficacy. The rA2DeltaNS1 and rA2DeltaM2-2 viruses were 2,200- to 55,000-fold restricted in replication in the upper and lower respiratory tracts but induced a level of RSV-neutralizing antibody in serum that was only slightly reduced compared to the level induced by wild-type RSV. The replication of wild-type RSV in immunized chimpanzees after challenge was reduced more than 10,000-fold at each site. Importantly, rA2DeltaNS1 and rA2DeltaM2-2 were 10-fold more restricted in replication in the upper respiratory tract than was the cpts248/404 virus, a vaccine candidate that retained mild reactogenicity in the upper respiratory tracts of 1-month-old infants. Thus, either rA2DeltaNS1 or rA2DeltaM2-2 might be appropriately attenuated for this age group, which is the major target population for an RSV vaccine. In addition, these results show that neither NS1 nor M2-2 is essential for RSV replication in vivo, although each is important for efficient replication.
Assuntos
Antígenos Virais/imunologia , Proteína HN , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/imunologia , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Animais , Antígenos Virais/genética , Mutação , Pan troglodytes , Recombinação Genética , Proteínas do Envelope Viral , Proteínas não Estruturais Virais/imunologia , Proteínas Virais/imunologiaRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the pathway for folic acid biosynthesis. The enzyme has been identified as a potential target for antimicrobial drugs. Equilibrium binding studies showed that Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue alpha, beta-methyleneadenosine triphosphate (AMPCPP) with high affinity. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP) exhibited a substantial fluorescence enhancement upon binding to HPPK, with an equilibrium dissociation constant comparable with that for ATP (10.4 and 4.5 micrometer, respectively). The apoenzyme did not bind the second substrate HMDP, however, unless AMPCPP was present, suggesting that the enzyme binds ATP first, followed by HMDP. Equilibrium titration of HPPK into HMDP and AMPCPP showed an enhancement of fluorescence from the pterin ring of the substrate, and a dissociation constant of 36 nm was deduced for HMDP binding to the HPPK.AMPCPP binary complex. Stopped flow fluorimetry measurements showed that the rate constants for the binding of MANT-ATP and AMPCPP to HPPK were relatively slow (3.9 x 10(5) and 1.05 x 10(5) m(-1) s(-1), respectively) compared with the on rate for binding of HMDP to the HPPK.AMPCPP binary complex. The significance of these results with respect to the crystal structures of HPPK is discussed.
Assuntos
Difosfotransferases/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Catálise , Fluorometria , Cinética , Ligantes , Modelos Químicos , ortoaminobenzoatos/metabolismoRESUMO
The M2 mRNA of human respiratory syncytial virus (RSV) contains two overlapping ORFs, encoding the transcription antitermination protein (M2-1) and the 90-aa M2-2 protein of unknown function. Viable recombinant RSV was recovered in which expression of M2-2 was ablated, identifying it as an accessory factor dispensable for growth in vitro. Virus lacking M2-2 grew less efficiently than did the wild-type parent in vitro, with titers that were reduced 1, 000-fold during the initial 2-5 days and 10-fold by days 7-8. Compared with wild-type virus, the intracellular accumulation of RNA by M2-2 knockout virus was reduced 3- to 4-fold or more for genomic RNA and increased 2- to 4-fold or more for mRNA. Synthesis of the F and G glycoproteins, the major RSV neutralization and protective antigens, was increased in proportion with that of mRNA. In cells infected with wild-type RSV, mRNA accumulation increased dramatically up to approximately 12-15 hr after infection and then leveled off, whereas accumulation continued to increase in cells infected with the M2-2 knockout viruses. These findings suggest that M2-2 mediates a regulatory "switch" from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly. With regard to vaccine development, the M2-2 knockout has a highly desirable phenotype in which virus growth is attenuated while gene expression is concomitantly increased.
Assuntos
Proteína HN , RNA Viral/biossíntese , Vírus Sincicial Respiratório Humano/genética , Transcrição Gênica , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Antígenos Virais/análise , Northern Blotting , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , RNA Mensageiro/análise , RNA Viral/análise , Vírus Sincicial Respiratório Humano/imunologia , Proteínas do Envelope Viral , Proteínas Virais/genéticaRESUMO
Using recombinant proteins extracted from mammalian cells, in a novel protein:protein binding assay, direct interaction of the nucleoprotein (NP) of simian virus 5 with the phosphoprotein (P) and V protein (V) was demonstrated. The amount of NP bound by V was found to be significantly less than that bound by P. Furthermore, preabsorption of NP with P removed the fraction of NP that could be bound by V, but preabsorption of NP with V did not remove all the NP that could be bound by P. These results suggested that V bound a subpopulation of the NP recognised by P. Further analysis revealed that P bound both soluble and homopolymeric forms of NP, while V bound only the soluble form; thus demonstrating that the binding sites on P and V, for soluble NP, are located within the N-terminal domain common to both P and V proteins. A monoclonal antibody, which recognised an epitope in the unique C-terminus of P, blocked the binding of P to polymeric NP but not to soluble NP. These results also suggest that there are two binding sites on NP for P, the site that interacts with the P/V common domain being either hidden or conformationally altered in polymeric NP.