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.

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.

Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays.

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.

Detection of influenza virus types A and B and type A subtypes (H1, H3, and H5) by multiplex polymerase chain reaction.

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.

Development of a TaqMan real-time RT-PCR assay for the detection of rabies virus.

Diagnosis of rabies relies on the fluorescent antibody test (FAT) from brain impression smears. The mouse brain inoculation test is used to confirm FAT but requires weeks until the result is known. TaqMan real-time PCR has been described for rabies viral RNA detection; however, this is burdened by primer and probe binding site mismatches. The purpose of this study was to develop a TaqMan real-time RT-PCR assay as an adjunct to FAT, based on national data of 239 rabies nucleoprotein sequences from rabies-infected brain specimens collected between 1998 and 2003. Two showed as many as 3 mismatches. However, mismatches on primer and/or probe binding sites did not affect amplification or detection. One hundred and forty-three brain samples submitted for rabies diagnosis from all over the country between 2005 and 2007 were also tested. Results were concordant with FAT. Thirteen rabies proven samples from Myanmar, Cambodia, Indonesia and India; 3 of which had up to 7 mismatches at primer/probe binding sites, could be detectable. Challenge Virus Standard, a fixed virus strain with 4 mismatches at probe binding site, could not be detected but remained amplified. This assay could be used as an adjunct to FAT and may serve as a rabies surveillance tool.

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.

Genetic characterization of H1N1, H1N2 and H3N2 swine influenza virus in Thailand.

Swine have been known to be a suitable host for influenza A virus. In Thailand, phylogenetic analysis on swine influenza virus (SIV) has as yet not been attempted. The present report presents molecular and phylogenetic analysis performed on SIV in Thailand. In this study, 12 SIV isolates from the central and eastern part of Thailand were subtyped and the molecular genetics of hemagglutinin and neuraminidase were elucidated. Three subtypes, H1N1, H1N2 and H3N2, are described. Phylogenetic analysis of the SIV hemagglutinin and neuraminidase genes shows individual clusters with swine, human or avian influenza virus at various global locations. Furthermore, amino acid substitutions were detected either at the receptor binding site or the antigenic sites of the hemagglutinin gene.