RESUMO
Highly pathogenic avian influenza (HPAI) H5N1 viruses have seriously affected the Asian poultry industry since their occurrence in 2004. Thailand has been one of those countries exposed to HPAI H5N1 outbreaks. This project was designed to compare the molecular evolution of HPAI H5N1 in Thailand between 2004 and 2008. Viruses with clade 1 hemagglutinin (HA) were first observed in early 2004 and persisted until 2008. Viruses with clade 2.3.4 HA were first observed in the northeastern region of Thailand between 2006 and 2007. Phylogenetic analysis among Thai isolates indicated that clade 1 viruses in Thailand consist of three distinct lineages: CUK2-like, PC168-like, and PC170-like viruses. The CUK2-like virus represents the predominant lineage and has been circulating throughout the course of the 4-year outbreaks. Analysis of recently isolated viruses has shown that the genetic distance was slightly different from viruses of the early outbreak and that CUK2-like viruses comprise the native strain. Between 2005 and 2007, PC168-like and PC170-like viruses were first observed in several areas around central and lower northern Thailand. In 2008, viruses reassorted from these two lineages, PC168-like and PC170-like viruses, were initially isolated in the lower northern provinces of Thailand and subsequently spread to the upper central part of Thailand. On the other hand, CUK2-like viruses were still detected around the lower northern and the upper central part of Thailand. Furthermore, upon emergence of the reassorted viruses, the PC168-like and PC170-like lineages could not be detected, suggesting that the only predominant strains still circulating in Thailand were CUK2-like and reassorted viruses. The substitution rate among clade 1 viruses in Thailand was lower. The virus being limited to the same area might explain the lower nucleotide substitution rate. This study has demonstrated that nationwide attempts to monitor the virus may help curb access and propagation of new HPAI viral genes.
Assuntos
Evolução Molecular , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Animais , Aves , Surtos de Doenças/veterinária , Hemaglutininas Virais/genética , Humanos , Influenza Aviária/epidemiologia , Mutação , Filogenia , Alinhamento de Sequência , Tailândia/epidemiologiaRESUMO
Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused several rounds of outbreaks in Thailand. In this study, we used 3 HPAI viruses isolated in Thailand in January 2004 from chicken, quail, and duck for genetic and pathogenetic studies. Sequence analysis of the entire genomes of these isolates revealed that they were genetically similar to each other. Chickens, quails, domestic ducks, and cross-bred ducks were inoculated with these isolates to evaluate their pathogenicity to different host species. A/chicken/Yamaguchi/7/04 (H5N1), an HPAI virus isolated in Japan, was also used in the chicken and quail studies for comparison. All four isolates were shown to be highly pathogenic to chickens and quails, with 100% mortality by 10(6) EID50 inoculants of the viruses. They caused sudden death in chickens and quails within 2-4 days after inoculation. The mean death times (MDT) of quails infected with the Thai isolates were shorter than those of chickens infected with the same isolates. Mortality against domestic and cross-bred ducks ranged from 50 to 75% by intranasal inoculation with the 10(6) EID50 viruses. Neurological symptoms were observed in most of the inoculated domestic ducks and appeared less severe in the cross-bred ducks. The MDTs of the ducks infected with the Thai isolates were 4.8-6 days post-inoculation. Most of the surviving ducks infected with the Thai isolates had sero-converted until 14 dpi. Our study illustrated the pathobiology of the Thai isolates against different poultry species and would provide useful information for improving control strategies against HPAI.
Assuntos
Galinhas , Patos , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/virologia , Codorniz , Administração Intranasal , Animais , Surtos de Doenças/veterinária , Genoma Viral , Testes de Inibição da Hemaglutinação/veterinária , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/imunologia , Influenza Aviária/mortalidade , Influenza Aviária/patologia , Reação em Cadeia da Polimerase/veterinária , RNA Viral/análise , Especificidade da Espécie , Organismos Livres de Patógenos Específicos , Tailândia/epidemiologia , Fatores de TempoRESUMO
In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.
Assuntos
Vírus da Influenza A/classificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Humanos , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/virologia , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
Infections with influenza virus type A and B present serious public health problems on a global scale. However, only influenza A virus has been reported to cause fatal pandemic in many species. To provide suitable clinical management and prevent further virus transmission, efficient and effective clinical diagnosis is essential. Therefore, we developed multiplex PCR assays for detecting influenza types A and B and the subtypes of influenza A virus (H1, H3 and H5). Upon performing multiplex PCR assays with type-specific primer sets, the clearly distinguishable products representing influenza A and B virus were separated by agarose gel electrophoresis. In addition, the subtypes of influenza A virus (H1, H3 and H5), which are most common in humans, can be readily distinguished by PCR with subtype-specific primer sets, yielding PCR products of different sizes depending on which subtype has been amplified. This method was tested on 46 influenza virus positive specimens of avian and mammalian (dog and human) origins collected between 2006 and 2008. The sensitivity of this method, tested against known concentrations of each type and subtype specific plasmid, was established to detect 10(3) copies/microl. The method's specificity was determined by testing against other subtypes of influenza A virus (H2, H4 and H6-H15) and respiratory pathogens commonly found in humans. None of them could be amplified, thus excluding cross reactivity. In conclusion, the multiplex PCR assays developed are advantageous as to rapidity, specificity, and cost effectiveness.
Assuntos
Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Vírus da Influenza A/metabolismo , Vírus da Influenza B/metabolismo , Reação em Cadeia da Polimerase/métodos , Animais , Análise Custo-Benefício , Primers do DNA/química , Cães , Eletroforese em Gel de Ágar , Humanos , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
This present study is the first to quantify the transmission of avian influenza virus H5N1 within flocks during the 2004 epidemic in Thailand. It uses the flock-level mortality data to estimate the transmission-rate parameter ( beta ) and the basic reproduction number (R(0)). The point estimates of beta varied from 2.26/day (95% confidence interval [CI], 2.01-2.55) for a 1-day infectious period to 0.66/day (95% CI, 0.50-0.87) for a 4-day infectious period, whereas the accompanying R(0) varied from 2.26 (95% CI, 2.01-2.55) to 2.64 (95% CI, 2.02-3.47). Although the point estimates of beta of backyard chickens and fighting cocks raised together were lower than those of laying hens and broiler chickens, this difference was not statistically significant. These results will enable us to assess the control measures in simulation studies. They also indicate that, for the elimination of the virus, a critical proportion of the susceptible poultry population in a flock (i.e., 80% of the population) needs to be vaccinated.
Assuntos
Galinhas/virologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/transmissão , Influenza Aviária/virologia , Animais , Surtos de Doenças , Mortalidade , TailândiaAssuntos
Galinhas/virologia , Surtos de Doenças/veterinária , Virus da Influenza A Subtipo H5N1/classificação , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Influenza Humana/epidemiologia , Amantadina/farmacologia , Animais , Antígenos Virais/genética , Antivirais/farmacologia , Genes Virais , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/genética , Masculino , Testes de Sensibilidade Microbiana , Oseltamivir/farmacocinética , Filogenia , Análise de Sequência , Especificidade da Espécie , Tailândia/epidemiologiaRESUMO
Avian influenza (AI) A virus subtypes H5 and H7 cause severe disease in domestic poultry, including chickens and turkeys. Moreover, H5 and H7 AI A viruses can cross the species barrier from poultry to humans. In the present study, we have developed a single-step multiplex reverse transcription-polymerase chain reaction assay (RT-PCR) for detecting H5 and H7 AI A viruses. This assay was applied to the poultry isolates with the aim of establishing a surveillance method to monitor possible transmission to humans. Two subtype-specific primer sets capable of producing PCR products of 157 and 326 base pairs corresponding to AI A virus H5 and H7 subtypes, respectively, were utilized in a one-step and one-tube reaction. The single-step multiplex RT-PCR assay developed in this study was found to be specific for detecting H5 and H7 AI A viruses. No specific amplification bands were detected with total nucleic acids extracted from other influenza hemagglutinin subtypes and other viral pathogens. The sensitivity of this assay was about 10(3) RNA copies/microl. In conclusion, this novel single-step multiplex RT-PCR is a simple assay with high potential for rapid, specific and cost effective laboratory diagnosis of H5 and H7 AI A virus isolates from clinical specimens of poultry.
Assuntos
Vírus da Influenza A/genética , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Aves/virologia , Eletroforese em Gel de Ágar , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H7N7/isolamento & purificação , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.
Assuntos
Antivirais/farmacologia , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Oseltamivir/farmacologia , Reação em Cadeia da Polimerase/métodos , Farmacorresistência Viral , Corantes Fluorescentes , Virus da Influenza A Subtipo H5N1/genética , Sensibilidade e EspecificidadeRESUMO
Three major outbreaks of avian influenza (AI) occurred in Thailand. During the third episode in October 2005, we have isolated H5N1 viruses from one human case and three poultry cases. The whole genomes of AI viruses from human, chickens and quail from the outbreaks were characterized. Sequence analysis of eight gene segments revealed that the 2005 H5N1 viruses isolated in October 2005 were closely related to those recovered from chicken, tiger(s) and human(s) in January and July 2004. In addition, the genetic changes of the AI isolates at the HA cleavage site have been observed.
Assuntos
Surtos de Doenças , Genoma Viral , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Animais , Surtos de Doenças/veterinária , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Influenza Aviária/epidemiologia , Dados de Sequência Molecular , Mutação , Filogenia , Aves Domésticas/virologia , Codorniz/virologia , Homologia de Sequência , Tailândia/epidemiologia , Tigres/virologiaAssuntos
Galinhas , Surtos de Doenças , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Influenza Aviária/virologia , Influenza Humana/virologia , Animais , Pré-Escolar , DNA Viral/química , DNA Viral/genética , Evolução Fatal , Hemaglutininas/química , Hemaglutininas/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Influenza Humana/patologia , Masculino , Neuraminidase/química , Neuraminidase/genética , Filogenia , Reação em Cadeia da Polimerase , Tailândia/epidemiologiaAssuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Desnaturação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , VirulênciaRESUMO
H5N1 influenza A virus causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to mammalian species such as leopards, tigers and humans. The aim of this study was to develop a multiplex real-time RT-PCR for rapid detection of H5N1 influenza A virus. The selected primers and various labeled TaqMan MGB reporter probes corresponding to M, H5 and N1 were used in a single step multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. In order to validate the method, 75 clinical specimens infected with H5N1 isolated from both poultry and mammals, as well as various specimens of other subtypes and RNA from other viral pathogens of poultry and human were tested. The results showed that the multiplex real-time RT-PCR assays can be applied to detect virus suspensions of H5N1 influenza A virus from a wide host range and demonstrated the sensitivity of the assay amounted to approximately 10(2)-10(3)copies/mul. In conclusion, the highlights of this particular method lie in its rapidity, specificity and sensitivity thus rendering it feasible and effective for large-scale screening at times of H5N1 influenza A virus outbreaks.
Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Aves , Primers do DNA , Fluorescência , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Dados de Sequência Molecular , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Panthera , Aves Domésticas , Sensibilidade e Especificidade , Proteínas da Matriz Viral/genéticaRESUMO
The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.
Assuntos
Animais de Zoológico/virologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Tigres/virologia , Sequência de Aminoácidos , Animais , Surtos de Doenças/veterinária , Humanos , Virus da Influenza A Subtipo H5N1/química , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tailândia/epidemiologia , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
In January 2004, highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype was first confirmed in poultry and humans in Thailand. Control measures, e.g., culling poultry flocks, restricting poultry movement, and improving hygiene, were implemented. Poultry populations in 1,417 villages in 60 of 76 provinces were affected in 2004. A total of 83% of infected flocks confirmed by laboratories were backyard chickens (56%) or ducks (27%). Outbreaks were concentrated in the Central, the southern part of the Northern, and Eastern Regions of Thailand, which are wetlands, water reservoirs, and dense poultry areas. More than 62 million birds were either killed by HPAI viruses or culled. H5N1 virus from poultry caused 17 human cases and 12 deaths in Thailand; a number of domestic cats, captive tigers, and leopards also died of the H5N1 virus. In 2005, the epidemic is ongoing in Thailand.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Aves Domésticas/virologia , Animais , Galinhas/virologia , Controle de Doenças Transmissíveis , Patos/virologia , Gansos/virologia , Humanos , Controle de Infecções/métodos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Influenza Humana/mortalidade , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Doenças das Aves Domésticas/prevenção & controle , Tailândia/epidemiologiaRESUMO
To determine whether avian H5N1 influenza viruses associated with human infections in Vietnam had transmitted to pigs, we investigated serologic evidence of exposure to H5N1 influenza virus in Vietnamese pigs in 2004. Of the 3,175 pig sera tested, 8 (0.25%) were positive for avian H5N1 influenza viruses isolated in 2004 by virus neutralization assay and Western blot analysis. Experimental studies of replication and transmissibility of the 2004 Asian H5N1 viruses in pigs revealed that all viruses tested replicated in the swine respiratory tract but none were transmitted to contact pigs. Virus titers from nasal swabs peaked on day 2, and low titers were detected in the liver of two of the four pigs tested. Our findings indicate that pigs can be infected with highly lethal Asian H5N1 viruses but that these viruses are not readily transmitted between pigs under experimental conditions.