Changes in the NS1 gene of avian influenza viruses isolated in Thailand affect expression of type I interferon in primary chicken embryonic fibroblast cells.

The non-structural protein 1 (NS1) of avian influenza virus was defined as one of the virulent factors. To understand the effect of NS1 protein of influenza virus H5N1 isolated in Thailand on type I (α/ß) interferon (IFN) synthesis, five reverse genetic viruses were constructed and used as models. The viruses were generated using NS genomic segment from A/Peurto Rico/8/1934 (H1N1) and four avian influenza viruses isolated from the first outbreak in Thailand. All the viruses have the rest of the genome from A/Peurto Rico/8/1934 (H1N1). The constructed viruses were named (1) NS1 PR8/34, (2) NS1 wild type, (3) NS1 L15FD53G, (4) NS1 N171I and (5) NS1 E71K, respectively. The type I (α/ß) IFN gene expression in control and infected primary chicken embryonic fibroblast cells were analyzed by quantitative polymerase chain reaction. The results show that the inhibition of IFN-ß gene expression by NS1 wild type infected cells is stronger than NS1 N171I, NS1 E71K, NS1 PR8/34 and NS1 L15FD53G, respectively. The data suggest that the difference of amino acid sequence of NS1 protein contributes to the IFN-ß antagonist. In contrast, the difference of the NS1 protein does not influence in the IFN-α antagonistic activity.

Detection of distribution of avian influenza H5N1 virus by immunohistochemistry, chromogenic in situ hybridization and real-time PCR techniques in experimentally infected chickens.

Ten specific pathogen free (SPF) chickens were inoculated intranasally with avian influenza virus subtype H5N1. Evaluation revealed distribution of the virus in twelve organs: liver, intestine, bursa, lung, trachea, thymus, heart, pancreas, brain, spleen, kidney, and esophagus. Immunohistochemistry (IHC), chromogenic in situ hybridization (CISH), and real-time polymerase chain reaction (PCR) were developed and compared for detection of the virus from the organs. The distribution of avian influenza H5N1 in chickens varied by animal and detecting technique. The heart, kidneys, intestines, lungs, and pancreas were positive with all three techniques, while the others varied by techique. The three techniques can be used to detect avian influenza effectively, but the pros and cons of each technique need to be determined. The decision of which technique to use depends on the objective of the examination, budget, type and quality of samples, laboratory facilities and technician skills.

Comparison of neuraminidase activity of influenza A virus subtype H5N1 and H1N1 using reverse genetics virus.

Neuraminidase (NA) is an envelope surface glycoprotein of influenza A viruses. It cleaves alpha-(2,3) or alpha-(2,6) glycosidic linkage between a terminal sialic acid residue of the host cell receptor and hemagglutinin of the viral envelope, thus releasing viral progeny from the infected cell. In this study, a reassortant virus (H1N1-NA-H5N1) containing the NA gene from A/duck/Phitsanulok/ NIAH6-5-0001/2007 (H5N1) virus and seven remaining genetic segments from A/ Puerto Rico/8/1934 (H1N1) was constructed using reverse genetic technique. NA activity of H1N1-NA-H5N1 virus was lower than that of A/Puerto Rico/8/1934 (H1N1), and NA activity of A/duck/Phitsanulok/NIAH6-5-0001/2007 study (H5N1) was the lowest among them (p < 0.05). To our knowledge, this is the first comparative study of NA activity of H1N1 and H5N1 virus using reverse genetic technique. It also indicates that the NA gene may be expressed at a higher level in the H1N1 infected cell than the H5N1 infected cell.

Genetic characterization of nonstructural genes of H5N1 avian influenza viruses isolated in Thailand in 2004-2005.

The outbreak of highly pathogenic avian influenza (HPAI) viruses has severely disrupted poultry production and trade. Humans have been infected with HPAI H5N1 viruses and many have died. The nonstructural (NS) proteins of the virus are a factor that determines virulence. In this report, 80 NS genes of H5N1 HPAI viruses isolated from Thailand were completely sequenced and phylogenically analyzed. The percentages of identity and variable site NS1 genes were similar to NS2/nuclear export protein (NEP) genes. All NS1 genes from the samples were located in allelic group A. The NS1 and NS2/NEP proteins possess 225 and 121 amino acids, respectively. All NS1 protein samples had five amino acid deletions typical of avian influenza viruses isolated since 2002. An amino acid substitution at position 92 (G92E) of the NS1 protein, known to promote the inhibition of host immune responses, was also found in the samples.

Whole genome sequences of H5N1 influenza a virus isolated from a little grebe in Thailand.

This is the first report of the whole genome sequence of influenza A virus in an aquatic resident bird of Thailand. It was categorized into genotype Z according to its characteristics of a 20 amino acid deletion in neuraminidase and a five amino acid deletion in the nonstructural protein. The indicator for a highly pathogenic trait of the virus is the presence of a polybasic amino acid sequence at the cleavage site of HA0. The feature of resistance to the antiviral drug amantadine is found at the 31st amino acid position of M2 (serine to asparagine). Phylogenic analyses revealed that virus A/little grebe/Thailand/Phichit-01/2004 (H5N1) is closely related to the chicken and human isolates recovered from Thailand. The high degrees of similarity among the sequences and phylogenic trees indicate there was no difference between the viruses isolated from poultry and aquatic birds in Thailand at the time of study. The results also suggest the source of H5N1 avian influenza virus in the little grebe and others in Thailand may have the same origin.