Influenza A and B in a cohort of outpatient children and adolescent with influenza like-illness during two consecutive influenza seasons

ABSTRACT Introduction Influenza is an important cause of morbimortality worldwide. Although people at the extremes of age have a greater risk of complications, influenza has been more frequently investigated in the elderly than in children, and inpatients than outpatients. Yearly vaccination with trivalent or quadrivalent vaccines is the main strategy to control influenza. Objectives Determine the clinical and molecular characteristics of influenza A and B infections in children and adolescents with influenza-like illness (ILI). Methods: A cohort of outpatient children and adolescents with ILI was followed for 20 months. Influenza was diagnosed with commercial multiplex PCR platforms. Results: 179 patients had 277 episodes of ILI, being 79 episodes of influenza A and 20 episodes of influenza B. Influenza A and B cases were mild and had similar presentation. Phylogenetic tree of influenza B viruses showed that 91.6% belonged to the B/Yamagata lineage, which is not included in trivalent vaccines. Conclusions: Influenza A and B are often detected in children and adolescents with ILI episodes, with similar and mild presentation in outpatients. The mismatch between the circulating influenza viruses and the trivalent vaccine offered in Brazil may have contributed to the high frequency of influenza A and B in this population.

Influenza A virus infection of healthy piglets in an abattoir in Brazil: animal-human interface and risk for interspecies transmission

Asymptomatic influenza virus infections in pigs are frequent and the lack of measures for controlling viral spread facilitates the circulation of different virus strains between pigs. The goal of this study was to demonstrate the circulation of influenza A virus strains among asymptomatic piglets in an abattoir in Brazil and discuss the potential public health impacts. Tracheal samples (n = 330) were collected from asymptomatic animals by a veterinarian that also performed visual lung tissue examinations. No slaughtered animals presented with any noticeable macroscopic signs of influenza infection following examination of lung tissues. Samples were then analysed by reverse transcription-polymerase chain reaction that resulted in the identification of 30 (9%) influenza A positive samples. The presence of asymptomatic pig infections suggested that these animals could facilitate virus dissemination and act as a source of infection for the herd, thereby enabling the emergence of influenza outbreaks associated with significant economic losses. Furthermore, the continuous exposure of the farm and abattoir workers to the virus increases the risk for interspecies transmission. Monitoring measures of swine influenza virus infections and vaccination and monitoring of employees for influenza infection should also be considered. In addition regulatory agencies should consider the public health ramifications regarding the potential zoonotic viral transmission between humans and pigs.

Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009.

Purpose: Influenza has a major impact on public heath, annually affecting 15-20% of the global population. Information on the activity of influenza virus in Mumbai is limited. The present study was carried out to determine the prevalence of influenza viruses causing acute respiratory infections in children by molecular methods. Objective: To study the prevalence of influenza viruses among the paediatric population in Mumbai by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Materials and Methods: From July 2007 to July 2009, 100 respiratory samples (nasal and throat swabs) were collected from paediatric patients with acute respiratory symptoms. attending out patients department, and admitted to the paediatric wards of B. J. Wadia Hospital for Children, Mumbai. The samples were collected and processed as per World Health Organization (WHO) guidelines. Viral RNA was extracted and one-step rRT-PCR was performed to detect influenza type A (H1 and H3) and influenza type B virus. Results: Out of 100 samples processed by rRT-PCR, a total of 11 samples (11%) were positive for influenza virus. The typing for influenza A subtypes showed 1% (1) positivity for H1 and 5% (5) positivity for H3 subtypes and 5% (5) samples tested positive for influenza type B virus. Conclusion: It was observed that both influenza type A and B viruses were prevalent in Mumbai during the study period. Such surveillance data are important in the early detection of any antigenic variants that may be helpful in global influenza vaccine preparation and for any pandemic preparedness activity.

Desenvolvimento de sistemas de genética reversa para os vírus da doença de gumboro e influenza aviária através do uso de recombinação homóloga em levedura / Development of reverse genetics systems for infectious bursal disease virus and avian influenza by yeast-based homologous recombination

A Doença de Gumboro (DG) é uma doença imunossupressora comum em aves jovens infectadas pelo Vírus da Doença de Gumboro (Infectious Bursal Disease Vírus, IBDV), sendo responsável por perdas econômicas no setor avícola. O vírus influenza apresenta-se com um alto nível de mutação, o que resulta no surgimento de vírus imunologicamente distintos capazes de causar pandemias ou epidemias. Entende-se por sistema de genética reversa viral (SGRV) a geração/recuperação de vírus por meio da transfecção celular do cDNA viral clonado ou seu RNA viral transcrito in vitro. SGRV pode ser usado na elucidação dos mecanismos de replicação do influenza e IBDV, e aplicações biotecnológicas como desenvolvimento de vacinas. Diante desse levantado, objetivou-se a construção de dois SGRVs por recombinação homóloga em levedura (RHL): um para IBDV e outro para influenza aviária (IA). Para o SGRV do IBDV, IBDV foi isolado no Brasil, teve seu genoma amplificado e clonado por RHL no vetor pJG-CMV-HDR. Os clones foram transfectados em fibroblasto de embrião de galinha (FEG) e o vírus gerado (IC-IBDVBr) mostrou estabilidade gênica e fenótipo similar ao vírus parental. A geração e crescimento do IC-IBDVBr não foram possíveis em células Vero. Para o SGRV do IA, IA foi isolado no Brasil, seu genoma foi amplificado e clonado em pDrive/pGEM-T Easy e depois subclonado por RHL no vetor pJGCh2008. Os clones em pJG-Ch2008 responsáveis pela codificação das proteínas do complexo polimerase viral (CPV) foram transfectados simultaneamente em células Human Embryonic Kidney 293T com plasmídeos contendo o gene repórter red fluorescent protein ou Gaussia luciferase, ambos flanqueados pela untransleated region do influenza. A funcionalidade do CPV do IA foi verificada pela expressão de RFP e GLuc. A recuperação do IA em FEG pelos clones em pJG-Ch2008 não foi possível. A funcionalidade do CPV mais a integridade dos clones indicam que a recuperação do IA não foi possível provavelmente devido à eficiência da transfecção celular. A construção do SGRV para IBDV, o primeiro do mundo feito por RHL e o primeiro desenvolvido no Brasil, junto com os passos iniciais para a construção do primeiro SGRV para influenza feito por RHL e a consequente construção do CPV por essa tecnologia, disponibilizam ao país ferramentas capazes de contribuir no esclarecimento do ciclo replicativo de ambos os vírus, além de criar bases para o futuro desenvolvimento de vacinas e vetores virais.

Leukocytoclastic Vasculitis Associated with Influenza A Virus Infection

Leukocytoclastic vasculitis (LCV) usually presents palpable purpura characterized by inflammation of vessel walls and fragmentation of nuclei. Various conditions can cause LCV, and it can be induced by influenza A virus infection. We report a 2-yr-old Korean girl who presented palpable purpuric and hemorrhagic lesions with fever. She was diagnosed as LCV by skin biopsy, and influenza A virus was isolated from nasopharyngeal swab. She was treated with oseltamivir (Tamiflu(R)) and prednisolone with dramatic effect of vasculitis and fever.

Virological surveillance and antiviral resistance of human influenza virus in Argentina, 2005–2008 / Vigilancia virológica y resistencia a los antivíricos del virus de la gripe humana en la Argentina, 2005–2008

Objective. To describe the virological characteristics of the influenza strains circulating in Argentina in 2005–2008 and to assess the prevalence of antiviral resistance. Methods. On the basis of their geographical spread and prevalence, influenza A and B isolates grown in Madin–Darby canine kidney cells were selected after antigenic and genomic characterization to be analyzed for antiviral resistance by enzymatic assay and pyrosequencing. Amantadine susceptibility was evaluated by pyrosequencing for known resistance markers on 45 strains of influenza A. Susceptibility to oseltamivir and zanamivir was evaluated by enzymatic assay of 67 influenza A and 46 influenza B strains, some of which were further analyzed by sequencing the neuraminidase gene. Results. Resistance to amantadine was observed only on A(H3N2) strains (29/33); all of them carried the mutation S31N in their M2 sequence. Oseltamivir resistance was observed in 12 (34.3%) of the 35 A(H1N1) strains from 2008; all of them carried the mutation H275Y in their neuraminidase sequence. All these viruses remained sensitive to zanamivir. Conclusions. This study describes a high incidence of amantadine-resistant influenza A(H3N2) viruses since 2006 and an unprecedented increase in oseltamivir resistance detected only in influenza A(H1N1) viruses isolated in 2008. Influenza A and B viruses were more sensitive to oseltamivir than to zanamivir, and influenza A viruses were more sensitive to both neuraminidase inhibitors than the influenza B viruses. The national data generated and analyzed in this study may help increase knowledge about influenza antiviral drug resistance, which is a problem of global concern.

Objetivo. Describir las características virológicas de las cepas de virus de la gripe que circulaban en la Argentina entre el 2005 y el 2008, y evaluar la prevalencia de la resistencia a los antivíricos. Métodos. Según su diseminación geográfica y su prevalencia, se seleccionaron aislados de gripe A y B cultivados en células renales caninas de Madin-Darby después de su caracterización antigénica y genómica, y se analizó su resistencia a los antivíricos mediante análisis enzimático y pirosecuenciación. La sensibilidad a la amantadina se evaluó por pirosecuenciación para los marcadores conocidos de resistencia en 45 cepas de gripe A. La sensibilidad al oseltamivir y al zanamivir se evaluó mediante análisis enzimático de 67 cepas de gripe A y 46 cepas de gripe B, algunas de las cuales se analizaron en mayor profundidad mediante la secuenciación del gen de la neuraminidasa. Resultados. Se observó resistencia a la amantadina solo en las cepas de gripe A (H3N2) (29/33); todas ellas tenían la mutación S31N en su secuencia de M2. Se observó resistencia al oseltamivir en 12 (34,3%) de las 35 cepas de gripe A (H1N1) aisladas en el 2008; todas ellas tenían la mutación H275Y en su secuencia de neuraminidasa. Todos estos virus conservaron su sensibilidad al zanamivir. Conclusiones. En este estudio se describe una incidencia elevada del virus de la gripe A (H3N2) resistente a la amantadina desde el 2006 y un aumento sin precedentes de la resistencia al oseltamivir detectada solo en los virus de la gripe A (H1N1) aislados en el 2008. Los virus de la gripe A y B fueron más sensibles al oseltamivir que al zanamivir y los virus de la gripe A fueron más sensibles a ambos inhibidores de la neuraminidasa que los virus de la gripe B. Los datos nacionales generados y analizados en este estudio pueden ayudar a aumentar los conocimientos acerca de la resistencia a los fármacos antivíricos dirigidos contra el virus de la gripe, lo que es un motivo de preocupación mundial.

Características del virus influenza y diagnóstico de laboratorio / Laboratory diagnosis and characterization of influenza virus

El virus influenza es causa frecuente de infección respiratoria. Pertenece a la familia Orthomixoviridae, existiendo tres géneros. Todos comparten características estructurales con un manto de lípidos y glicoproteínas hemaglutinina y neuraminidasa, que participan en la patogenicidad viral y determinando los diferentes subtipos de virus; en su interior una hebra de ácido ribonucleico de polaridad negativa. El presente artículo resumen las principales características de laboratorio, clínicas y de diagnóstico de este emergente virus respiratorio.

Multiplex reverse transcription-PCR assay for detection of type A influenza virus plus differentiation of avian H7 and H9 hemagglutinin subtypes in Iran

Avian influenza virus [AIV] infection is a major cause of bird and human morbidity and mortality. We aimed to evaluate a specific and sensitive multiplex RT-PCR that can simultaneously detect influenza type A viruses and differentiate the two most important subtypes of avian influenza viruses H7 and H9 subtypes. A multiplex reverse transcriptase-polymerase chain reaction [mRT-PCR] was developed and optimized for the detection of type A influenza virus. Simultaneously avian H7 and H9 hemagglutinin subtypes was differentiated. Three sets of specific oligonucleotide primers were used in this test for type A influenza virus, H7 and H9 heamagglutinin subtypes. The mRT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 313 bp for H7 and 428 bp for H9 hemagglutinin subtypes, and 101 bp for type A influenza virus. The common set of primers for type A influenza virus were able to amplify a 101 bp DNA band for any of the other subtypes of influenza A virus The mRT-PCR assay developed in this study was found to be sensitive and specific. No specific amplification bands of the same sizes [313 and 428 bp] could be amplified for RNA of other influenza hemagglutinin subtypes, nor specific amplification bands of type A influenza [101 bp] for Influenza B, C, or other viral or bacterial pathogens tested in this study

Occurrence of swine influenza virus infection in swine with porcine respiratory disease complex.

We studied the occurrence of swine influenza virus (SIV) infection in piglets with respiratory symptoms resembling porcine respiratory disease complex (PRDC). A total of 106 samples including nasal swab and lung suspension from sick piglets were collected from 30 farms of medium size in the central and eastern parts of Thailand from August 2006 to February 2007. Samples were inoculated onto Mardin-Darby Canine Kidney (MDCK) cells and SIV infection was confirmed by immunofluorescent assay (IFA) and reverse transcriptase polymerase chain reaction (RT-PCR) specific for M gene. Of 106 samples, 3 pigs from 3 different farms were found to be SIV positive on all assays. The positive samples were further identified by RT-PCR as H3N2 subtype using specific primers for hemagglutinin (HA) and neuraminidase (NA) genes. SIV infection was found in 2.8% of swine suffering from respiratory distress suggesting SIV may not be the major pathogen for PRDC in the central and eastern Thailand. SIV was present in 3 of 30 farms (10%) indicating the prevalence of SIV in these regions is considerable. Since pigs are vulnerable to infection from both human and avian influenza viruses and interspecies transmission between humans and swine occurs sporadically, it is essential to continue surveillance and monitoring of SIV infection in the swine population.

Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestations / Vírus influenza e bactéria proteolítica co-infectantes em trato respiratório de indivíduos com manifestações respiratórias

A role for proteolytic bacteria in the exacerbation of influenza virus has been shown in natural hosts such as pigs and humans. Four hundred seven samples were collected from the respiratory tract of individuals presenting clinical manifestations, during influenza season (2003-2005) in São Paulo City. The aim of this study was to evaluate the incidence of determined bacteria co-infecting virus in human respiratory tract. Tests, such as bacteriological, immunofluorescence (IF), RT/PCR and hemagglutination (HA) were used for bacterial and viral investigation. Thirty seven (9.09 percent) positive for influenza virus were screened by IF. The RT/PCR confirmed the presence of influenza virus in these samples. Bacterial and agar casein tests demonstrated that 18 (48.64 percent) individuals were infected with proteolytic bacteria such as Staphylococcus spp., Streptococcus spp. and Pseudomonas spp. Among these samples, 13 (35.13 percent) were co-infected with influenza A virus. Influenza type B, co-infecting bacteria were found in five (13.51 percent) samples. In vitro the S. aureus protease increased the influenza HA titer after contact for 30 min at 25 ºC. Results revealed the occurrence of co-infection with proteolytic bacteria and influenza in the evaluated individuals. This finding corroborates that virus versus bacteria synergism could be able to potentiate respiratory infection, increasing damage to hosts.

O papel da bactéria proteolítica na exacerbação do vírus influenza tem sido demonstrado em hospedeiros naturais como porcos e humanos. Foram coletadas 407 amostras do trato respiratório de indivíduos apresentando manifestações clínicas, durante a estação da influenza (2003-2005) na cidade de São Paulo. Este trabalho teve como objetivo avaliar a incidência de determinadas bactérias que junto com vírus co-infectarem o trato respiratório humano. Testes bacteriológicos, e virológicos como imunofluorescência (IF), RT/PCR e hemaglutinação (HA) foram usados nas investigações viral e bacteriana. Pelo teste de IF foram selecionadas trinta e sete (9,09 por cento) amostras positivas para o vírus influenza. A presença do vírus influenza foi confirmada pela técnica de RT/PCR. Pelos testes bacteriológicos e do agar caseina, verificou-se que 18 (48,64 por cento) dos indivíduos foram infectados com bactérias proteolíticas tais como Staphylococcus spp., Streptococcus spp. e Pseudomonas spp. Destas amostras, 13 (35,13 por cento) foram co-infectadas com vírus influenza tipo A, e 5 (13,51 por cento) com influenza tipo B. No experimento in vitro com influenza e S. aureus, detectou-se aumento do título hemaglutinante deste vírus, após contacto de 30 min a 25 ºC. Os resultados obtidos revelaram a ocorrência de co-infecção com bactéria proteolítica e vírus influenza nos indivíduos avaliados. Estes achados corroboram com a investigação do sinergismo, entre bactéria e vírus, que poderia ser capaz de potencializar infecção respiratória, aumentando os riscos aos hospedeiros.

bird flu: a new emerging pandemic threat and its pharmacological intervention

Bird flu is an infection caused by avian influenza viruses, which are of different types A, B and C. Type A avian influenza viruses are the most frequently associated with avian influenza epidemics and pandemics. There are 16 hemagglutinin [H1 to H16] and 9 neuraminidase types [N1 to N9] identified till date. A peculiar characteristic of influenza A viruses is their propensity for genetic change by two main processes: antigenic drift [small, gradual changes] and antigenic shift [abrupt, major change producing a novel influenza A virus subtype]. There are various modes of transmission of human influenza including inhalation, direct or indirect [fomite] contact etc., can have manifestations ranging from mild to severe or fatal disease, depend on the viral subtype causing the disease. Avian influenza A [H5N1] results in high death rate amongst infants and young children. The first outbreak of human infection by avian influenza viruses [H5N1] was observed in 1997 in Hong Kong. Since then a large number of outbreaks have been reported in different parts of the world. In fact, the spread of avian influenza H5N1 in various species including humans has lead to a current pandemic threat. Human avian influenza infections in persons at high risk of exposure can be prevented by adopting a series of protective measures, anti-viral vaccination and health monitoring. Drugs currently available for the treatment or prophylaxis of influenza infections include the adamantanes [amantadine and rimantadine] and the newer class of neuraminidase inhibitors [zanamivir, oseltamivir and peramivir]. However, vaccines are considered the first line of defense for reducing the excess morbidity and mortality that invariably accompany pandemics and a number of clinical trials are under way to test them

La influenza, un problema vigente de salud pública / Influenza, an existing public health problem

La influenza estacional es una enfermedad respiratoria aguda, recurrente y común que se conoce desde la antigüedad y se presenta sobre todo durante los meses de invierno con un elevado impacto para la salud pública mundial. La enfermedad se manifiesta con altas tasas de morbilidad en individuos de todas las edades y elevadas tasas de mortalidad en niños, individuos mayores de 60 años, pacientes con enfermedades crónicas y mujeres en gestación. Las estrategias de prevención incluyen el uso de vacunas: inactivadas, subunitarias o vacuna con virus genéticamente modificados. Dos subtipos de virus de influenza tipo A y un virus de influenza tipo B causan la enfermedad en humanos. Los virus de influenza A que afectan a los humanos mutan con facilidad, por lo que con frecuencia aparecen nuevas variantes antigénicas de cada subtipo, lo que obliga a incluir dichas variantes en las vacunas anuales para brindar una adecuada protección a la población. La influenza pandémica se refiere a la introducción y posterior diseminación mundial de un nuevo virus de influenza en la población humana, lo que ocurre de manera esporádica, y que debido a que los humanos carecen de inmunidad para el nuevo virus pueden suscitarse epidemias graves con elevadas tasas de morbilidad y mortalidad. Históricamente el origen de las pandemias de influenza se debe a la transmisión de virus de aves al hombre o la transferencia de genes de éstos a los virus de la influenza estacional. En las aves acuáticas silvestres, tanto migratorias como costeras, se mantiene una gran diversidad de subtipos de virus de influenza, los cuales se introducen eventualmente en aves domésticas, donde algunos virus adquieren la capacidad de infectar a mamíferos, incluido el hombre. El proceso de adaptación de los virus aviarios a hospederos mamíferos requiere tiempo, por lo que la presentación de estos casos puede tardar varios años. Desde diciembre de 2003, en varios países del sureste asiático, las aves domésticas han sido afectadas por una epidemia de influenza aviaria (subtipo H5N1) de grandes proporciones. A febrero de 2006 la epidemia ya afectó a países de Europa y Africa, con un fuerte impacto económico para la avicultura comercial por el sacrificio de más de 180 millones de aves. Algunos linajes de este virus adquirieron la capacidad de cruzar la barrera de especie e infectaron de manera directa pero incipiente a la población humana...

Isolation, sequencing and phylogenetic analysis of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 and H3N8

We report here on the isolation and sequencing of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 (A⁄equi-1⁄Santiago⁄77, Sa77) and H3N8 (A⁄equi-2⁄Santiago⁄85, Sa85) . The sequences obtained allowed a variability analysis, which indicated significant differences when compared with other isolates. We found that Chilean isolates are more similar to the North American variety than to European isolates. Isolate Sa77 is a good candidate for inclusion in a vaccine as it is the latest isolate of the subtype H7N7 and is probably better-adapted to the equine host. Isolate Sa85, of subtype H3N8, also appears to be a good candidate since it has no significant differences in the main antigenic sites with recent isolates.

Isolation of the new variant influenza A (H3N2) strains during an outbreak in Pune, February, 1996.

During January-February, 1996, an outbreak of influenza-like illness occurred in Pune. The throat and nasal swabs collected from the patients during this outbreak were processed in MDCK and LLC-MK2 cell cultures and influenza A(H3N2) viruses were isolated. They were identified as being similar to the recent circulating global strains A/Johannesburg/33/94 and A/Wuhan/359/95.

Hemagglutinating and sialidase activities of subpopulations of influenza A viruses

The present study was conducted to investigate the characteristics of two samples of influenza A/England/42/72 (H3N2) virus, one of them selected by an adsorption-elution technique, to determine the possible existence of virus variants or subpopulations. Based on specificity of virulence-related cell receptor-binding and sialidase activities, this selection technique using human O group erythrocytes revealed the presence of variants within a standard virus sample with diversity for their hemagglutinating and sialidase activities. The standard-like (E1) sample exhibited titers of 4 and 32 HAU (hemagglutinating units in 25 microliters) with human O group and chicken erythrocytes, respectively, while the sample obtained by the adsorption-elution process (E2) exhibited titers of 32 and 4 HAU, respectively, with these same types of erythrocytes. The E2 sample showed higher sialidase activity at pH values between 5.4 and 6.6 with human erythrocytes (128-256 HAU), but the E1 sample did not exhibit significant sialidase activity with either human or chicken erythrocytes. The different pH optima for hemolysis (5.2) and sialidase (5.4-6.6) activities and the higher hemolysis indexes present in samples with sialidase activity inhibited by heating (at 56 degrees C for 30 min) or by treatment with EDTA (dilution in buffer containing 2 mM EDTA, a chelating agent on calcium-dependent sialidase activity) demonstrate the independence of these activities in the selected sample: native E2 (absorbance = 0.18), EDTA-treated native E2 (absorbance = 0.28), heated E2 (absorbance = 0.26), EDTA-treated heated E2 (absorbance = 0.41).(ABSTRACT TRUNCATED AT 250 WORDS)