The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand

In this report, the genome of the Thai avian influenza virus A (H5N1); A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04, isolated from the Thai avian influenza A (AI) epidemic during the early of 2004 was sequenced. Phylogenetic analyses were performed in comparison to AI viruses from Hong Kong 1997 outbreaks and other AI (H5N1) isolates reported during 2001-2004. Molecular characterization of the Thai AI (H5N1) HA gene revealed a common characteristic of a highly pathogenic AI (HPAI), a 20-codon deletion in the neuraminidase gene, a 5-codon deletion in the NS gene and polymorphisms of the M2 and PB2 genes. Moreover, the HA and NA genes of the Thai AI displayed high similarity to those of the AI viruses isolated from human cases during the same epidemic. Finally, our results demonstrated that the Thai AI emerged as a member of 2000's AI lineage with most of the genetic sequences closely related to the Influenza A/Duck/China/E319.2/03 (H5N1).     

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.     

Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong.

Analysis of the sequences of all eight RNA segments of the influenza A/G oose/Guangdong/1/96 (H5N1) virus, isolated from a sick goose during an outbreak in Guangdong province, China, in 1996, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5N1 viruses isolated in Hong Kong in 1997. However, the remaining genes showed greater similarity to other avian influenza viruses. Notably, the neuraminidase gene did no have the 19-amino-acid deletion in the stalk region seen in the H5N1 Hong Kong viruses and the NS gene belonged to allele B, while that of the H5N1 Hong Kong viruses belonged to allele A. These data suggest that the H5N1 viruses isolated from the Hong Kong outbreaks derived their HA genes from a virus similar to the A/Goose/Guangdong/1/96 virus or shared a progenitor with this goose pathogen.     

Genetic analysis of H9N2 avian influenza viruses isolated from India

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003-2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R-S-S-R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R-S-N-R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.     

Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004

The first known cases of human infection with highly pathogenic avian influenza (HPAI) H5N1 viruses in Vietnam occurred in late 2003. However, HPAI H5N1 and low-pathogenic avian influenza (LPAI) H5N2 and H9N3 viruses were isolated from domestic waterfowl during live-bird market (LBM) surveillance in Vietnam in 2001 and 2003. To understand the possible role of these early viruses in the genesis of H5N1 strains infecting people, we performed sequencing and molecular characterization. Phylogenetic analysis revealed that the hemagglutinin (HA) genes of two geese HPAI H5N1 strains belonged to clade 3, and their surface glycoprotein and replication complex genes were most closely related (98.5–99.7% homologous) to A/duck/Guangxi/22/01 (H5N1) virus, detected contemporarily in southern China, whilst the M and NS genes were derived from an A/duck/Hong Kong/2986.1/00 (H5N1)-like virus. The H5 HA gene of the duck HPAI H5N1 strain belonged to clade 5 and acquired a gene constellation from A/quail/Shantou/3846/02 (H5N1), A/teal/China/2978.1/02 (H5N1) and A/partridge/Shantou/2286/03 (H5N1)-like viruses. The phylogenetic analysis further indicated that all eight gene segments of goose and duck HPAI H5N1 and LPAI H5N2 viruses were distinct from those of H5N1 clade-1 viruses known to have caused fatal human infections in Vietnam since late 2003. The duck H9N3 isolates derived genes from aquatic-bird influenza viruses, and their H9 HA belonged to the Korean lineage. The PB2 gene of A/duck/Vietnam/340/01 (H9N3) virus had lysine at position 627. Based on the molecular characterization of specific amino acid residues in the surface and relevant internal protein-coding genes, the Vietnamese H5N1 and H9N3 virus isolates indicated specificity to avian cell surface receptor and susceptibility for currently licensed anti-influenza A virus chemotherapeutics. Our findings suggest that the H5N1 and H5N2 viruses that circulated among geese and ducks in LBMs in Hanoi, Vietnam, during 2001 and 2003 were not the immediate ancestors of the clade-1 viruses associated with fatal human infections in Vietnam. The clade-1 HPAI H5N1 viruses were independently introduced into Vietnam.     

Characterization of a highly pathogenic H5N1 avian influenza virus isolated from ducks in Eastern China in 2011

This study describes the characterization of seven H5N1 avian influenza viruses from domestic ducks in Eastern China in 2011. Phylogenetic analysis showed these viruses were closely related to an H5N1 virus circulating in wild birds in Hong Kong. Some characteristics of these viruses were similar to those of an H5N1 strain that circulated in China and Vietnam (2003-2004). The virulence of three isolates was examined in chickens and mice, and they were found to be highly pathogenic in chickens but showed low pathogenicity in mice. These results suggest that continued H5N1 surveillance in poultry should be used as an early warning system for avian influenza outbreaks.     

Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia

The reported transmission of avian H9N2 influenza viruses to humans and the isolation of these viruses from Hong Kong poultry markets lend urgency to studies of their ecology and pathogenicity. We found that H9N2 viruses from North America differ from those of Asia. The North American viruses, which infect primarily domestic turkeys, replicated poorly in inoculated chickens. Phylogenetic analysis of the hemagglutinin and nucleoprotein genes indicated that the Asian H9N2 influenza viruses could be divided into three sublineages. Initial biological characterization of at least one virus from each lineage was done in animals. Early isolates of one lineage (A/Chicken/Beijing/1/94, H9N2) caused as high as 80% mortality rates in inoculated chickens, whereas all other strains were nonpathogenic. Sequence analysis showed that some isolates, including the pathogenic isolate, had one additional basic amino acid (A-R/K-S-S-R-) at the hemagglutinin cleavage site. Later isolates of the same lineage (A/Chicken/Hong Kong/G9/97, H9N2) that contains the PB1 and PB2 genes similar to Hong Kong/97 H5N1 viruses replicated in chickens, ducks, mice, and pigs but were pathogenic only in mice. A/Quail/Hong Kong/G1/97 (H9N2), from a second lineage that possesses the replicative complex similar to Hong Kong/97 H5N1 virus, replicated in chickens and ducks without producing disease signs, was pathogenic in mice, and spread to the brain without adaptation. Examples of the third Asian H9N2 sublineage (A/Chicken/Korea/323/96, Duck/Hong Kong/Y439/97) replicated in chickens, ducks, and mice without producing disease signs. The available evidence supports the notion of differences in pathogenicity of H9N2 viruses in the different lineages and suggests that viruses possessing genome segments similar to 1997 H5N1-like viruses are potentially pathogenic in mammals.     

Molecular aspects of avian influenza (H5N1) viruses isolated from humans

In 1997, 18 human infections with H5N1 influenza type A were identified in Hong Kong and six of the patients died. There were concomitant outbreaks of H5N1 infections in poultry. The gene segments of the human H5N1 viruses were derived from avian influenza A viruses and not from circulating human influenza A viruses. In 1999 two cases of human infections caused by avian H9N2 virus were also identified in Hong Kong. These events established that avian influenza viruses can infect humans without passage through an intermediate host and without acquiring gene segments from human influenza viruses. The likely origin of the H5N1 viruses has been deduced from molecular analysis of these and other viruses isolated from the region. The gene sequences of the H5N1 viruses were analysed in order to identify the molecular basis for the ability of these avian viruses to infect humans.     

Detection of Hong Kong 97-like H5N1 influenza viruses from eggs of Vietnamese waterfowl

Summary. Three H5N1 influenza viruses were isolated from shell washes of duck and goose eggs confiscated from travelers coming from Vietnam. All eight gene segments of these viruses share high sequence identity with the H5N1 avian influenza viruses that caused outbreaks in poultry and humans in Hong Kong in 1997. Animal studies indicate that these isolated viruses are able to replicate in mouse lung and could be found in the organs of ducks without causing any clinical signs or death. However, the viruses are highly pathogenic for chickens. Although the source of these recently isolated Hong Kong 97-like H5N1 viruses is undetermined, their detection in the egg shell of duck and goose suggests that this particular genotype of H5N1 virus may have re-emerged in nature or may have been circulating continuously.     

Potency of an inactivated influenza vaccine prepared from A/duck/Hong Kong/960/1980 (H6N2) against a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice

H6 influenza viruses are prevalent in domestic and wild birds in Eurasian countries and have been isolated from pigs and a human. To prepare for an influenza pandemic, we have established an influenza virus library consisting of more than 1,300 influenza virus strains, including 144 combinations of 16 hemagglutinin and 9 neuraminidase subtypes. H6 viruses in the library were classified into Early, Group II, Group III, and W312 sublineages and the North America lineage on the basis of their phylogenetic features. Chicken antisera to A/duck/Hong Kong/960/1980 (H6N2) of the Early sublineage broadly reacted with viruses of different sublineages in a hemagglutinin inhibition test. A whole inactivated virus particle vaccine was prepared from A/duck/Hong Kong/960/1980 (H6N2) which was stocked in the influenza virus library. The potency of this vaccine against A/duck/Vietnam/OIE-0033/2012 (H6N2), which belongs to a different sublineage, was evaluated in mice. The test vaccine was sufficiently potent to induce an immune response that reduced the impact of disease caused by a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice. The present results indicate that the whole inactivated virus particle vaccine prepared from a virus strain in the influenza virus library is useful as a vaccine against pandemic influenza.     

Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003

Avian H9N2 influenza A virus has caused repeated human infections in Asia since 1998. Here we report that an H9N2 influenza virus infected a 5-year-old child in Hong Kong in 2003. To identify the possible source of the infection, the human isolate and other H9N2 influenza viruses isolated from Hong Kong poultry markets from January to October 2003 were genetically and antigenically characterized. The findings of this study show that the human H9N2 influenza virus, A/Hong Kong/2108/03, is of purely avian origin and is closely related to some viruses circulating in poultry in the markets of Hong Kong. The continued presence of H9N2 influenza viruses in poultry markets in southern China increases the likelihood of avian-to-human interspecies transmission.     

Pandemic threat posed by avian influenza A viruses

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.     

Characterization of a pathogenic H9N2 influenza A virus isolated from central China in 2007

The entire genome of the A/Chicken/Hubei/C1/2007 (H9N2) virus, isolated from central China in 2007, was completely sequenced and phylogenetically analyzed. Phylogenetic analysis demonstrated that A/Chicken/Hubei/C1/2007 (H9N2) virus represents multiple reassortant lineages, with genes coming from the early mainland China strain (Ck/Beijing/1/94), an H9N2 virus with special genotype (Ck/shanghai/F/98) and other lineages from poultry in Asia. Infection studies indicated that A/Chicken/Hubei/C1/2007 (H9N2) virus replicated efficiently in MDCK cells and in BALB/c mice. The H9N2 virus also replicated to high titers in chicken respiratory tracts and caused overt clinical signs in chickens. Our results suggest that attention should be paid to the natural evolution of H9N2 influenza viruses and to the control of H9N2 influenza viruses in animals.     

Genome characterisation of the newly discovered avian influenza A H5N7 virus subtype combination

Summary. In Denmark, in 2003, a previously unknown subtype combination of avian influenza A virus, H5N7 (A/Mallard/Denmark/64650/03), was isolated from a flock of 12,000 mallards. The H5N7 subtype combination might be a reassortant between recent European avian influenza A H5, H7, and a third subtype, possibly an H6. The haemagglutinin and the acidic polymerase genes of the virus were closely related to a low-pathogenic Danish H5N2 virus A/Duck/Denmark/65041/04 (H5N2). The neuraminidase gene and the non-structural gene were most similar to the highly pathogenic A/Chicken/Netherlands/1/03 (H7N7) and the human-fatal A/Netherlands/219/03 (H7N7), respectively. The basic polymerase 1 and 2 genes were phylogenetically equidistant to both A/Duck/Denmark/65047/04 (H5N2) and A/Chicken/Netherlands/1/03 (H7N7). The nucleoprotein and matrix gene had highest nucleotide sequence similarity to the H6 subtypes A/Duck/Hong Kong/3096/99 (H6N2) and A/WDk/ST/1737/2000 (H6N8), respectively. All genes of the H5N7 strain were of avian origin, and no further evidence of pathogenicity to humans has been found.     

Characterization of H5N1 influenza A viruses isolated during the 2003-2004 influenza outbreaks in Japan

In Japan, between the end of December 2003 and March 2004, four outbreaks of acute, highly transmissible and lethal disease occurred in birds in three prefectures separated by 150-450 km, involving three chicken farms and a group of chickens raised as pets. The cause of each outbreak was an H5N1 influenza A virus-the first highly pathogenic virus to be isolated from the outbreaks in Japan since 1925. The H5N1 virus was also isolated from dead crows, apparently infected by contact with virus-contaminated material. These H5N1 viruses were antigenically similar to each other, but could be differentiated from other H5 viruses, including those isolated from Hong Kong in 1997 and 2003, by use of a panel of monoclonal antibodies in hemagglutination inhibition assays. Genetically, the H5N1 viruses in Japan were closely related to each other in all genes and were genetically closely related to a single isolate of genotype V that was isolated in 2003 in the Guandong Province of mainland China (A/chicken/Shantou/4231/2003). The virulence of the index isolate (A/chicken/Yamaguchi/7/2004) was studied in chickens and mice. Chickens intravenously or intranasally inoculated with the isolate died within 1 or 3 days of inoculation, respectively. In mice, although this virus replicated well in the lung without prior adaptation and spread to the brain, the dose lethal to 50% of the mice was 5 x 10(5) 50% egg infectious doses (EID50), which is less pathogenic than the Hong Kong 1997 H5N1 viruses isolated from humans. Our findings indicate that the H5N1 viruses associated with the influenza outbreaks in chickens in Japan were genotypically closely related to an H5N1 virus isolated from chicken in China in 2003 (genotype V), but were different from those prevalent in southeastern Asia in 2003-2004 (i.e., genotype Z) and that these highly pathogenic viruses can be transmitted to crows, which are highly susceptible to these viruses.     

我国猪群中H9N2亚型毒株HA和NA基因特性的研究

目的 了解我国内地从猪中分离到H9N2亚型毒株HA和NA基因来源及它们使猪致病的原因。方法 用PCR扩增目的基因，与P^GEM-T Easy Vector4℃过夜连接，重组质粒转化DH-10β细菌，筛选阳性菌落，酶切鉴定，测序。然后，进行进化树分析。结果 两株猪H9N2毒株HA蛋白分子上第226位上氨基酸为L，这与从人和猪所分离出的H9N2毒株相同，其连接肽属对禽致病的毒株，但它们的序列为R-L-S-R，而不是R-S-S-R；其NA蛋白茎区第62～64位存在掉失，这与A／Shaoguarn/408／98，A／Swine／Hong Kong／9／98及A／Duck／Hong Kong／y280／97(H9N2)毒株相同；HA与NA基因进化树分析表明，两株猪H9N2毒株的HA基因接近于A／Chicken／Hong Kong／G23／97和A／Chicken／Hong Kong／G9／97．而NA基因接近于A／Shaoguan／408／98毒株。结论 两株猪H9N2亚型毒株的HA和NA基因可能性最大来自禽H9N2毒株。由于其HA蛋白分子上连接肽氨基酸序列发生替换，可能造成了它们对猪具有致病性。禽H9N2毒株NA蛋白茎区氨基酸掉失，造成了它们能直接感染猪。     

Influenza viruses and their ion channels

Influenza is an ancient disease that has infected humans in irregular intervals throughout recorded history. The most infamous pandemic was "Spanish Flu" which affected large parts of the world population and killed in 1918-1919, at a rough estimate, at least 50 million people. More recently, two influenza A pandemics occurred in 1957 ("Asian influenza") and 1968 ("Hong Kong influenza") and caused significant morbidity and mortality globally. Most recently, in 1997 and 2003, limited outbreaks caused by a new influenza A virus subtype H5N1 that was directly transmitted from birds to humans, occurred in the Hong Kong Special Administrative Region of China. Since 2003, the avian H5N1 strain has infected more then 130 persons in Vietnam, Thailand, and Cambodia and has killed more than half of them. To prevent the human and economical losses caused by human and avian influenza viruses, it is necessary to prepare safe universal influenza vaccines. In order to develop a broad-spectrum protection against different influenza virus strains or variants, some recent studies have were aimed at the M2 protein of Influenza A virus. This review compares the influenza A, B, and C viruses by focusing on their ion channels.     

Potency of an inactivated avian influenza vaccine prepared from a non-pathogenic H5N1 reassortant virus generated between isolates from migratory ducks in Asia

A reassortant influenza virus, A/duck/Hokkaido/Vac-1/2004 (H5N1) (Dk/Vac-1/04), was generated between non-pathogenic avian influenza viruses isolated from migratory ducks in Asia. Dk/Vac-1/04 (H5N1) virus particles propagated in embryonated chicken eggs were inactivated with formalin and adjuvanted with mineral oil to form a water-in-oil emulsion. The resulting vaccine was injected intramuscularly into chickens. The chickens were challenged with either of the highly pathogenic avian influenza virus strains A/chicken/Yamaguchi/7/2004 (H5N1) or A/swan/Mongolia/3/2005 (H5N1) at 21 days post-vaccination (p. v.), when the geometric mean serum HI titers of the birds was 64 with the challenge virus strains. The vaccinated chickens were protected from manifestation of disease signs upon challenge with either of the highly pathogenic avian influenza viruses. However, challenge virus was recovered at low titers from the birds at 2 and 4 days post-challenge (p.c.). All 3 chickens challenged at 6 days p.v. died, whereas 3 chickens challenged at 8 days p.v. survived. These results indicate that the present vaccine confers clinical protection and reduction of virus shedding against highly pathogenic avian influenza virus challenge and should be useful as an optional tool in emergency cases.     

Pathogenicity and antigenicity of a new influenza A (H5N1) virus isolated from duck meat

Avian influenza A viruses are the ancestral origin of all human influenza viruses. The outbreak of highly pathogenic (HP) avian H5N1 in Hong Kong in 1997 highlighted the potential of these viruses to infect and cause severe disease in humans. Since 1999, HP H5N1 viruses were isolated several times from domestic poultry in Asia. In 2001, a HP H5N1 virus, A/Duck/Anyang/AVL-1/2001 (Dk/Anyang), was isolated from imported frozen duck meat in Korea. Because of this novel source of HP H5N1 virus isolation, concerns were raised about the potential for human exposure and infection; we therefore compared the Dk/Anyang virus with HP H5N1 viruses isolated from humans in 1997 in terms of antigenicity and pathogenicity for mammals. At high doses, Dk/Anyang virus caused up to 50% mortality in BALB/c mice, was isolated from the brains and lymphoid organs of mice, and caused lymphopenia. Overall Dk/Anyang virus was substantially less pathogenic for mice than the H5N1 virus isolated from a fatal human case in 1997. Likewise, Dk/Anyang virus was apathogenic for ferrets. Dk/Anyang virus was antigenically distinguishable by hemagglutination-inhibition (HI) assay from human H5N1 viruses isolated in 1997 and avian H5N1 viruses isolated in 2001 in Hong Kong. Nevertheless, prior infection with Dk/Anyang virus protected mice from death after secondary infection with HP human H5N1 viruses. These results indicate that compared with HP human H5N1 viruses, Dk/Anyang virus is substantially less pathogenic for mammalian species. Nevertheless, the novel source of isolation of this avian H5N1 virus must be considered when evaluating the potential risk to public health.     

Characterization of a highly pathogenic avian influenza H5N1 virus sublineage in poultry seized at ports of entry into Vietnam

Highly pathogenic avian influenza H5N1 virus was detected in poultry seized at two ports of entry located in Lang Son Province, Vietnam. Sequence analysis of the hemagglutinin (HA) genes from five H5N1 virus isolates and ten PCR amplicons from chicken cloacal samples revealed their close phylogenetic relationship to clade 7 H5N1 HA genes. However, these HA genes exhibited extensive genetic divergence at both the nucleotide and amino acid levels in comparison to previously described clade 7 viruses; e.g., A/chicken/Shanxi/2/2006. In addition, hemagglutination inhibition tests revealed antigenic differences between these and previously isolated H5N1 viruses from Vietnam. These results indicate that viruses with clade 7 HA are evolving rapidly in poultry in Southeast Asia.