RESUMO
The antigenic properties of H3N8 influenza viruses isolated from outbreaks of equine influenza in Sweden between 1979 and 1991 have been studied in hemagglutination inhibition tests with polyclonal and monoclonal antisera, and antigenic drift of the virus has been demonstrated. To clarify the basis of the antigenic drift, amino acid sequences of the globular head regions (HA1) of the hemagglutinin membrane glycoproteins of virus strains from 1979, 1984, 1988 and 1990 have been deduced from the nucleotide sequences of the hemagglutinin genes, and the sequence information has been used to construct a phylogenetic tree of H3N8 equine influenza strains. Several strains from previous studies have been included to give a clearer picture of viral evolution in an international context.
Assuntos
Evolução Biológica , Hemaglutininas Virais/genética , Doenças dos Cavalos/virologia , Vírus da Influenza A Subtipo H3N8 , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/veterinária , Sequência de Aminoácidos , Animais , Anticorpos , Anticorpos Monoclonais , Sequência de Bases , Frequência do Gene , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Cavalos , Vírus da Influenza A/isolamento & purificação , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/virologia , RNA Viral/isolamento & purificação , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Suécia , Fatores de Tempo , Proteínas do Envelope Viral/genéticaRESUMO
The antigenic properties of H3N8 equine influenza virus from the Swedish epizootic of 1991 differ from those of A/eq 2/Fontainebleau/79 (representative of the Swedish vaccine strain) in hemagglutination inhibition tests. The amino acid sequence of the hemagglutinin (HA) of an isolate from the 1991 outbreak was deduced from the nucleotide sequence and comparison was made to the A/eq 2/Fontainebleau/79 strain. Twenty-three amino acid substitutions were found, 10 mapping onto areas of the HA known to bind antibodies in human H3 influenza viruses. The amino acid changes together with the serological data suggest that a major antigenic drift has taken place in equine H3N8 viruses in Sweden and we conclude that recent strains of the virus must be incorporated into vaccines on a regular basis if epizootics of equine influenza are to be controlled in the future.
Assuntos
Antígenos Virais/genética , Variação Genética , Vírus da Influenza A/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/imunologia , Sequência de Bases , Embrião de Galinha , DNA Viral , Surtos de Doenças , Epitopos , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Doenças dos Cavalos/epidemiologia , Cavalos , Vírus da Influenza A/imunologia , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Homologia de Sequência de Aminoácidos , Suécia/epidemiologiaRESUMO
In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza virus (A2). We show that the assays are specific for the target genes chosen, and display sensitivity similar to virus isolation. The NP assay detects a variety of different influenza subtypes, whereas A1 and A2 assays are specific for influenza subtypes H7N7 and H3N8, respectively. Sequencing of amplicons obtained in the A2 assay yields information on antigenic regions of the haemagglutinin molecule, and use of this procedure in the routine surveillance of equine influenza will enable tentative characterisation of circulating viruses despite difficulties in isolating field strains of the H3N8 subtype. The A1 assay will be useful in ascertaining whether viruses of the H7N7 subtype still circulate amongst horses, or whether these are extinct.
Assuntos
Doenças dos Cavalos/diagnóstico , Vírus da Influenza A/isolamento & purificação , Doenças Nasais/veterinária , Infecções por Orthomyxoviridae/veterinária , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Embrião de Galinha , Primers do DNA/química , DNA Viral/química , Eletroforese em Gel de Ágar , Imunofluorescência/veterinária , Testes de Inibição da Hemaglutinação/veterinária , Testes de Hemaglutinação/veterinária , Doenças dos Cavalos/virologia , Cavalos , Vírus da Influenza A/classificação , Doenças Nasais/diagnóstico , Doenças Nasais/virologia , Infecções por Orthomyxoviridae/diagnóstico , Reação em Cadeia da Polimerase/veterinária , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sensibilidade e Especificidade , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Direct amplification and sequencing of the hemagglutinin (HA) genes of equine influenza virus subtype H3N8 was undertaken in order to characterize strains of this virus circulating in Sweden. The majority of viruses from outbreaks during 1997 analyzed belonged to the American lineage of H3 equine influenza, and one strain was shown to belong to the European lineage. Furthermore, it was shown that recent American-lineage strains are mutated at amino acid position 190 of the HA during serial passage in embryonated hens' eggs. Host cell adaptation of these viruses thus takes place at antigenic region B of the HA.
Assuntos
Vírus da Influenza A Subtipo H3N8 , Vírus da Influenza A/classificação , Sequência de Aminoácidos , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Cavalos , Vírus da Influenza A/genética , Dados de Sequência Molecular , FilogeniaRESUMO
Four Swedish strains of equine H3N8 influenza virus isolated from outbreaks during the last 4 years were characterized. Antigenic typing using monoclonal antibodies raised against a variety of H3N8 strains showed that the viruses are heterogeneous, the 1993 isolate being closely related to the 1991 Swedish isolate TAB/91 and the other three isolates from 1994 and 1996 being more closely related to each other. This pattern is reflected in the phylogenetic data calculated from nucleotide sequencing of the haemagglutinin genes. H3N8 equine influenza can be seen to be evolving in two distinct lineages, one European and one American. The 1993 isolate is closely related to the European lineage and is the most recent Swedish strain of this lineage to be isolated. The 1994 and 1996 isolates fit into the American lineage, which contains recent isolates from the United States and also Britain. These results indicate that American-type H3N8 viruses have become endemic in Sweden and, in light of the antigenic differences which can be observed between viruses belonging to the two lineages, we believe that equine influenza virus vaccines should be updated with an American-type virus strain.