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Background: Influenza A viruses are capable of crossing the specific barrier between human beings and animals resulting in interspecies transmission. The important factor of potential infectivity of influenza A viruses is the suitability of the receptor binding site of the host and viruses. The affinities of avian and human influenza virus to bind with the receptors and the distributions of receptors in animals are different., Objective: This study aims to investigate the anatomical distribution of avian and human influenza virus receptors using the double staining lectin histochemistry method., Methods: Double staining of lectin histochemistry was performed to identify both SA alpha2,3 Gal and SA alpha2,6 Gal receptors in trachea and lung tissue of dogs, cats, tigers, ferret, pigs, ducks and chickens., Results: We have demonstrated that avian and human influenza virus receptors were abundantly present in trachea, bronchus and bronchiole, but in alveoli of dogs, cats and tigers showed SA alpha2,6 Gal only. Furthermore, endothelial cells in lung tissues showed presence of SA alpha2,3 Gal., Conclusion: The positive sites of both receptors in respiratory tract, especially in the trachea, suggest that all mammalian species studied can be infected with avian influenza virus. These findings suggested that dogs and cats in close contact with humans should be of greater concern as an intermediate host for avian influenza A in which there is the potential for viral adaptation and reassortment.

The highly pathogenic avian influenza virus H5N1 is known to induce high level of tumor necrosis factor alpha (TNF-alpha) from primary macrophages. However, it is still unclear whether current H5N1 strains also induce high TNF-alpha production, as most of the data were derived from extinct clade 0 H5N1 strain. Here, we show that current clade 1 and 2 H5N1 strains induce variable levels of TNF-alpha that are not necessarily higher than those induced by seasonal influenza viruses. The result suggests that hyper-induction of TNF-alpha in human macrophages is not always associated with a highly pathogenic phenotype. We further tested the contribution of the NS gene segment from H5N1 isolates to TNF-alpha induction by using reverse genetics. While NS conferred some variation in TNF-alpha induction when incorporated into an H1N1 virus genetic background, it did not affect TNF-alpha induction in an H5N1 virus genetic background, suggesting that other viral genes are involved.

A double-antigen sandwich ELISA was developed for the detection of antibodies to influenza A viruses. A recombinant nucleoprotein (rNP) of influenza A virus was used as a capture antigen and an HRP-conjugate for detecting the antibodies. A total of 125 serum samples from birds of different species including chickens, geese, open-billed storks, Khaki Campbell ducks, lesser whistling ducks, and pigeons with known antibodies were tested by ELISA. The sensitivity and the specificity of ELISA were found to be 98% and 97.3%, respectively. The assay was able to detect the presence of influenza A antibodies as early as the fourth day post-inoculation in ducks infected experimentally with influenza A (H5N1) virus. Excellent agreement (97.6%) was obtained between this sandwich ELISA and the hemagglutination inhibition (HI) tests (kappa=0.95). The double-antigen sandwich ELISA correlated well with a commercial avian influenza (AI) multispecies ELISA and was slightly more sensitive than the AI multispecies ELISA. These findings indicate that the double-antigen sandwich ELISA based on rNP may offer an effective screening method for serodiagnosis of influenza A virus. The double-antigen sandwich ELISA also enables the detection of antibodies to influenza A viruses in different species without the need for species-specific secondary antibodies., (2009 Elsevier B.V. All rights reserved.)

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.

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.

House sparrows, European starlings, and Carneux pigeons were inoculated with 4 influenza A (H5N1) viruses isolated from different avian species. We monitored viral replication, death after infection, and transmission to uninfected contact birds of the same species. Sparrows were susceptible to severe infection; 66%-100% of birds died within 4-7 days. High levels of virus were detected from oropharyngeal and cloacal swabs and in organs of deceased sparrows. Inoculation of starlings caused no deaths, despite high levels of virus shedding evident in oropharyngeal swabs. Least susceptible were pigeons, which had no deaths and very low levels of virus in oropharyngeal and cloacal swabs. Transmission to contact birds did not occur frequently: only A/common magpie/Hong Kong/645/2006 virus was shown to transmit to 1 starling. In summary, recent influenza (H5N1) viruses are pathogenic for small terrestrial bird species but the rate of intraspecies transmission in these hosts is very low.

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) occurred in various types of domestic poultry in Thailand during 2004-05. H5N1 viruses were also detected in humans and other mammalian species. Infections were mainly detected in backyard chickens and domestic ducks. The geographic distribution of the 2004 outbreaks was widespread throughout Thailand; most outbreaks occurred in the Central Region, the southern part of the Northern Region, and the Eastern Region. In 2005, the H5N1 outbreaks continued and showed a clustered pattern in four provinces in the southern part of the Northern Region and in one province in the Central Region. H5N1 HPAI outbreaks caused serious socioeconomic consequences to the poultry industry, the social community, farmers' livelihood, and human health. After key measures were implemented, the incidence of the outbreaks declined remarkably in 2005.

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.

H5N1 avian influenza viruses are continuing to spread in waterfowl in Eurasia and to threaten the health of avian and mammalian species. The possibility that highly pathogenic (HP) H5N1 avian influenza is now endemic in both domestic and migratory birds in Eurasia makes it unlikely that culling alone will control H5N1 influenza. Because ducks are not uniformly killed by HP H5N1 viruses, they are considered a major contributor to virus spread. Here, we describe a reverse genetics-derived high-growth H5N3 strain containing the modified H5 of A/chicken/Vietnam/C58/04, the N3 of A/duck/Germany/1215/73, and the internal genes of A/PR/8/34. One or two doses of inactivated oil emulsion vaccine containing 0.015 to 1.2 microg of HA protein provide highly efficacious protection against lethal H5N1 challenge in ducks; only the two dose regimen has so far been tested in chickens with high protective efficacy.

In addition to causing 12 human deaths and 17 cases of human infection, the 2004 outbreak of H5N1 influenza virus in Thailand resulted in the death or slaughter of 60 million domestic fowl and the disruption of poultry production and trade. After domestic ducks were recognized as silent carriers of H5N1 influenza virus, government teams went into every village to cull flocks in which virus was detected; these team efforts markedly reduced H5N1 infection. Here we examine the pathobiology and epidemiology of H5N1 influenza virus in the 4 systems of duck raising used in Thailand in 2004. No influenza viruses were detected in ducks raised in "closed" houses with high biosecurity. However, H5N1 influenza virus was prevalent among ducks raised in "open" houses, free-ranging (grazing) ducks, and backyard ducks.

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.

During the second outbreak of avian influenza H5N1 in Thailand, probable horizontal transmission among tigers was demonstrated in the tiger zoo. Sequencing and phylogenetic analysis of those viruses showed no differences from the first isolate obtained in January 2004. This finding has implications for influenza virus epidemiology and pathogenicity in mammals.