Overview of avian influenza virus in urban feral pigeons in Bangkok, Thailand.

This survey assessed the presence of avian influenza virus (AIV) in urban feral pigeons (UFPs) in Bangkok, Thailand. A total of 485 UFPs were collected from eight study sites, and blood, tracheal, and cloacal samples were collected from each bird. Virus isolation and molecular methods did not detect AIV in any of the birds tested. A hemagglutination inhibition test was used to test for antibodies to high and low pathogenicity AIV subtypes. AIV subtype H9 antibodies were the only antibodies detected. The overall seroprevalence of AIV subtype H9 antibodies was 6.9%, and subtype H9 antibodies were found in UFPs at all eight sites. The overall geometric mean titer was 11.07 (range: 8-64). These results reveal that UFPs in Bangkok do not currently pose a risk of transmitting AIV to humans. However, monitoring of AIV in UFPs is necessary for disease control and to minimize the possibility of influenza outbreaks.

Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species.

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.

Surveillance activities and molecular analysis of H5N1 highly pathogenic avian influenza viruses from Thailand, 2004-2005.

Avian influenza (AI) outbreaks were first reported in Thailand in January 2004. In the past 2 yr, AI viruses have caused three epidemic waves. Disease prevention and control in all aspects have been actively carried out. Active and passive surveillance based on clinical observation and laboratory analysis were intensively conducted, as well as monitoring of genetic variation of the viruses. H5N1 viruses isolated from different avian species from different cases and locations were selected. We have sequenced specific genes (HA, NA, M, Ns, and part of PB2 genes) of 58 H5N1 isolates, as well as whole genome sequencing of 21 Thai influenza A (H5N1) viruses isolated during the 2004-2005 outbreak. Cluster analysis study showed that AI isolates were identified as highly pathogenic avian influenza (HPAI) and belonged to genotype Z. The virus had a multiple basic amino acid motif at the cleavage site of HA, deletions in the NA stalk region, a five amino acid deletion in the NS1 gene, and genetic markers for amantadine resistance in the M2 gene. All 58 H5N1 isolates were closely related and grouped into the same cluster, together with isolates from wild birds, cats, tigers, and humans. Phylogenetic analysis also revealed that Thai isolates were in the same cluster as Vietnamese isolates but aligned in a different cluster from Indonesian, Hong Kong, and Chinese viruses. In addition, genetic analysis showed that most avian influenza virus (AIV) isolates from Thailand had no major genetic changes in each gene such as HA (HA cleavage site, receptor binding site, N-link glycosylation site), NA (NA stalk region, oseltamivir resistance marker), M (the amantadine resistance marker, host specificity site), NS (five amino acid deletion site), and PB2 (host specificity site). All Thai poultry isolates contained the amantadine resistance marker while none of them had the oseltamivir resistance marker. To this end, the molecular characterization of H5N1 viruses from Thailand showed that there were no significant point mutations in the critical regions, and there was no evidence of changes in the viruses that indicate they are capable of sustained human-to-human transmission.

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand.

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.

Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection.

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.

Highly pathogenic avian influenza H5N1, Thailand, 2004.

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.

Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004.

The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.

The out of Africa model of varicella-zoster virus evolution: single nucleotide polymorphisms and private alleles distinguish Asian clades from European/North American clades.

Until 1998, varicella-zoster virus (VZV) was generally considered sufficiently stable to allow the use of a single sequenced virus (VZV-Dumas) as a consensual representation of the world VZV genotype. But recent investigations have uncovered a gE mutant virus called VZV-MSP with a second genotype and a distinguishable accelerated cell spread phenotype. A subsequent study suggested that single nucleotide polymorphisms (SNPs) could be applied toward the genetic analysis of the VZV genome. To further assess the scope of genetic variation in the VZV genome on a worldwide basis, we carried out an extensive SNP analysis of structural glycoprotein genes gB, gE, gH, gI, gL, as well as the IE62 regulatory gene in viruses collected from Western Europe, North America and Asia, including the VZV vaccine strain. The SNP data showed segregation of viral isolates of Asian origin from those of Western ancestry into distinct phylogenetic clades. Unexpectedly, however, VZV from Thailand segregated with VZV from Iceland and the United States, i.e. it was more Western than Asian in nature. Further, SNP analysis disclosed strikingly unusual genotypes, e.g. gH genes with up to five missense mutations and gL genes with insertions of an in-frame methionine codon. In summary, these VZV genomic analyses have shown that individual VZV strains, like closely related human beings, have distinctive SNP profiles containing private alleles within just five VZV genes (gB, gH, gE, gL and IE62) that provide a fingerprint to localize ancestry of the viral strain.