Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSRdownward arrowGLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSRdownward arrowGLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.

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.

Epidemiological studies on highly pathogenic avian influenza H5N1 during late 2009 and 2010 in Egypt

Epidemiological studies on AI virus H5N1 in different governorates in Egypt [Alexandria, Bohera, Cairo, El-fayom, Gharbia, Giza and Dakahlia] during late 2009 and 2010 were carried out. These studies included seventy five flocks [49 broilers, 17 layer, 4 breeder and 5 duck flocks] showing high mortality rate with respiratory manifestations and diarrhea, through isolation in specific pathogen free eggs and identification by hemagglutination inhibition test using H5N1 antiserum. The data revealed the following: a percentage of 32.7%, 76.5%, 50% and 40% in broiler, layer, breeder and duck flocks respectively were positive for AIV H5N1. The highest incidence was recorded in layer flocks followed by broiler breeder flocks, duck then broiler flocks with total incidence of 44% in all species. Sequencing and phylogenetic tree of hemagglutinin [HA] gene of six positive AIV H5N1 isolates from chickens during 2010 were done. Phylogenetic tree showed that all HA gene sequences blonged to highly diverse clade 2.2.1 viruses according to WHO/FAO/OIE nomenclature. Analysis of amino acid sequences of HA glycoprotein revealed some mutations at the receptor binding site of the HA molecule

Factors affecting hemagglutination activity of avian influenza virus subtype H5N1

Hemagglutination [HA] and hemagglutination inhibition [HI] tests for avian influenza [AI] virus [H5N1] were standardized using varying various factors like erythrocytes from different species, type of diluent, incubation temperature and incubation period. The virus was peropagated in embryonated chicken eggs [9-11 days]. The allantoic fluid [AF] was harvested 36 hours post incubation and was confirmed by slide hemagglutination test. The maximum HA titres were obtained using 1% RBCs of chicken, sheep, duck, geese, pigeon, quails and turkey for 30-40 minutes. The haemagglutination activity showed best titer when used phosphate buffer saline than normal saline and also when incubated at 25[degree sign] C instead of 37[degree sign] C

Teoría de la escalera térmica / Thermic stairways theory

In this article we raise the possibility of a potential relationship between body temperature of different spe-cies of animáis and the Avian Flu. We hypothesize that the different valúes of body temperature could act as an enabling mechanism for Influenza virus infection through an adaptative mechanism of the virions in the species susceptible to infection, including human beings.

En el presente artículo se plantea una posible relación entre la temperatura corporal de diferentes especies animales y la presentación de la Gripe aviar. Se postula que los diferentes valores de temperatura corporal podrían actuar como un mecanismo favorecedor de las infecciones por virus Influenza, mediante un mecanismo de tipo ad-aptativo de los viriones en las diversas especies animales susceptibles a la infección, incluyendo al humano.

Influenza aviar / Avian influenza

Se hace referencia a las características virológicas de la influenza aviar, expansión geográfica y migración de las aves en los Continentes, probabilidad de riesgo de pandemia en un futuro cercano, descripción clínica de la patología y terapéutica disponible, así como también a los avances preventivos y recomendaciones.

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...

Influenza humana y aviaria: pasado, presente y futuro / Avian and human influenza: past, present and future

Se revisa la epidemiología de la influenza en los últimos años y el brote en curso de influenza aviaria y sus riesgos para el hombre. En el siglo pasado hubo tres pandemias graves de influenza humana causadas por distintos subtipos de virus A que se extendieron por todo el mundo. Según las evidencias clínicas, de laboratorio y epidemiológicas disponibles, existe un riesgo real de una nueva pandemia que eventualmente afectaría al hombre derivada de la epidemia aviaria, A/H5N1, cuyo principal reservorio son aves migratorias. Se inició en Hong Kong en el año 2003 propagándose con gran rapidez a la población avícola en varios países de Asia, Rusia, Macedonia, Colombia y Turquía. Chile no ha sido afectado. La trasmisión ave-hombre está documentada en un número reducido de casos pero se teme que el virus, mediante recombinación genética con cepas humanas, adquiera la capacidad de infectar y trasmitirse al hombre dando curso a su diseminación explosiva. Se discute la estrategia, indicaciones y limitaciones de su prevención y tratamiento incluyendo vigilancia epidemiológica, empleo de fármacos antivirales e inmunización.

efficacy of inactivated oil-emulsion H9N2 avian influenza vaccine

An experimental inactivated oil-emulsion H9N2 avian influenza vaccine was formulated with 3 parts of inactivated avian influenza antigen A/Chicken/lran/101/1998[H9N2] emulsified in 7 parts of oil adjuvant. Twelve week-old specific pathogen-free [SPF] chickens were divided into seven groups of 10 birds. Six groups were vaccinated with 1, 1/10th, 1/50th, 1/100th, 1/200th and 1/400th field dose of the experimental avian influenza vaccine [EAIV]. The last group, was injected with saline and served as the control group. The mean titer in haemagglutination inhibition [HI] test [log 2] on the vaccinated groups, 21 days post-vaccination were 6.0, 4.4, 3.83, 3.3, 3.0 and 2, respectively. Prevention of virus shedding through cloaca was used as the potency test which revealed that the protective doses 50% [PD50] of full, 1/10th and 1/50th of the field dose of the experimental vaccine were 100, 100 and 96.25%, respectively. Those groups that received <1/50th dose could not prevent virus shedding. So it can be concluded that EAI vaccine could even be entirely protective and efficient in 1/10th dose and got a desirable immunity in experimental SPF chickens

serological survey for detection of avian infectious bronchitis virus antibodies in domestic village chickens in Esfahan, central Iran

Infectious bronchitis [1B] is a very contagious disease caused by a coronavirus [IBV]. In chickens, the virus affects the respiratory, reproductive, and urinary systems. This study was carried out to determine the seroprevalence of anti-IBV antibodies in domestic village chickens. Serum samples of 300 domestic village chickens from Esfahan [centeral Iran] were collected and examined for the presence of anti-lBV antibodies by commercial ELISA kits. The results showed that 85.3% of the domestic village chickens had high titers of anti-IBV antibody without any clinical signs. It was concluded that the rate of IBV infection in these chickens is very high that could be a potential hazard for commercial poultry