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.     

Antigenic, genetic, and pathogenic characterization of H5N1 highly pathogenic avian influenza viruses isolated from dead whooper swans (Cygnus cygnus) found in northern Japan in 2008

In April and May 2008, whooper swans (Cygnus cygnus) were found dead in Hokkaido in Japan. In this study, an adult whooper swan found dead beside Lake Saroma was pathologically examined and the identified H5N1 influenza virus isolates were genetically and antigenically analyzed. Pathological findings indicate that the swan died of severe congestive edema in the lungs. Phylogenetic analysis of the HA genes of the isolates revealed that they are the progeny viruses of isolates from poultry and wild birds in China, Russia, Korea, and Hong Kong. Antigenic analyses indicated that the viruses are distinguished from the H5N1 viruses isolated from wild birds and poultry before 2007. The chickens vaccinated with A/duck/Hokkaido/Vac-1/2004 (H5N1) survived for 14 days after challenge with A/whooper swan/Hokkaido/1/2008 (H5N1), although a small amount of the challenge virus was recovered from the tissues of the birds. These findings indicate that H5N1 highly pathogenic avian influenza viruses are circulating in wild birds in addition to domestic poultry in Asia and exhibit antigenic variation that may be due to vaccination.     

Vaccination of chickens against H5N1 avian influenza in the face of an outbreak interrupts virus transmission.

Vaccination of chickens with a commercially available killed H5N2 vaccine was being evaluated as an additional tool to enhanced biosecurity measures and intensive surveillance for control of highly pathogenic avian influenza subtype H5N1 disease in Hong Kong in 2002. In December 2002 to January 2003, there were outbreaks of H5N1 disease in waterfowl in two recreational parks, wild water birds, several poultry markets and five chicken farms. In addition to quarantine, depopulation of the affected sheds and increased biosecurity, vaccination of the unaffected sheds and surrounding unvaccinated farms was undertaken on three farms. In at least two farms, infection spread to the recently vaccinated sheds with low rates of H5N1 mortality in sheds when the chickens were between 9 and 18 days post-vaccination. However, after 18 days post-vaccination no more deaths from H5N1 avian influenza occurred and intensive monitoring by virus culture on these farms showed no evidence of asymptomatic shedding of the virus. This provides evidence that H5 vaccine can interrupt virus transmission in a field setting.     

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.     

Pathogenicity of a highly pathogenic avian influenza virus, A/chicken/Yamaguchi/7/04 (H5N1) in different species of birds and mammals

Summary. Outbreaks of highly pathogenic avian influenza (HPAI) have been occurring in domestic poultry in Asia since 1996. In the beginning of 2004, HPAI outbreaks were caused by H5N1 virus in two farms and a group of pet chickens in different areas of Japan. In the present study, the pathogenicity of A/chicken/Yamaguchi/7/04 (H5N1), which had been isolated from a dead chicken during the first outbreak in Japan, was assessed in chickens, quails, budgerigars, ducklings, mice, and miniature pigs by experimental infection. The virus was highly pathogenic to all the birds tested. Mice were susceptible to infection with a low mortality rate and miniature pigs were resistant to infection with the virus.     

Isolation of influenza A/H5N1 virus strains from poultry and wild birds in West Siberia during epizooty (July 2005) and their depositing to the state collection of viruses (August 8, 2005)

Six strains of influenza AH5N1 virus were isolated, by using PS and MDCK continuous cell lines from poultry and wild birds, which were collected on July 28, 2005 in the samples taken from 5 examines species of wild birds in the Novosibirsk region during the epizootic outbreak with a high mortality. The strains were identified by means of HIT, RT-PCR, and microchip-based techniques. Two strains, A/Grebe/Novosibirsk/29/05 (H5N1) and A/Duck/Novosibirsk/56/05 (H5N1), were deposited to the Russian State Collection of Viruses (Registration Nos. 2372 and 2371, respectively) with the priority date 08.08.2005. Positive results in RT-PCR for influenza A/H5N1 virus detec- tion were obtained in 100% of the samples from dead and sick poultry; 93% from the clinically healthy poultry kept together with sick one; positive results ranged from 0 to 36%. Sequencing established the identity of genetic characteristics of strains isolated for wild birds and poultry as well as their affiliation to high pathogenic avian influenza (HPAI). Phylogenetic analysis revealed a high homology of hemagglutining of West-Siberian strains and strains isoolated from bar-headed gooses (Eulabeia indica) on the Qinghai Lake (Western China) in the 2005 spring.     

Genome-sequenee Analysis of the Pathogenic H5N1 Avian Influenza A Virus Isolated in China in 2004

Analysis of the sequences of the genome of the avian influenza A/chicken/Hubei/327/2004 (H5N1) virus, isolated from a poultry farm during the outbreak of avian influenza (AI) in Hubei Province, central China, in the spring of 2004, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5 highly pathogenic avian influenza virus (HPAI). Notably, the neuraminidase gene of the virus had a 20-amino acid deletion in the stalk region and a 5-amino acid deletion in the NS gene which belonged to allele B. Furthermore, the internal genes (PB2, PA, NP, M2) of the A/chicken/Hubei/327/2004 virus with the particular amino acid residues were more closely related to H5N1 viruses of 2000–2003 isolated in Hong Kong and the AIV of Thailand and Vietnam in 2004, but less likely to evolve from the viruses of Hong Kong 1997. Finally, our results demonstrated that the influenza A/chicken/Hubei/327/2004 (H5N1) virus was similar to those of the AI viruses isolated from Hong Kong (2000–2003), Vietnam, and Thailand rather than the viruses from the 1997 lineage of Hong Kong and with closest genetic relatives to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus. These data suggest that the influenza A/chicken/Hubei/327/2004 (H5N1) virus which circulated in central China derived its internal gene from a virus similar to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus.     

Double introduction of highly pathogenic H5N1 avian influenza virus into France in early 2006.

Highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 have spread since late 2003 in East and Southeast Asia. In April 2005, a large-scale outbreak of H5N1 infection that occurred in migratory waterfowl in Qinghai Lake nature reserve in western China, killing more than 6000 wild birds, appeared to be the beginning of a epizootic that caused outbreaks in domestic and wild birds in nearly 60 countries from Central Asia, the Middle East, Europe and Africa. The first case of Asian lineage HPAI H5N1 virus in France was described in dead wild ducks (Common pochard) in the east of France in mid-February 2006. Up to the end of April, 42 HPAI H5N1 viruses were identified from about 60 wild birds belonging to different species and one outbreak occurred in commercial turkeys. To establish genetic relationships with other HPAI H5N1 viruses, 12 selected viruses were subjected to phylogenetic analysis. Genotyping and genetic analyses revealed that the French viruses were very similar to those of the 'Qinghai-like' sublineage and belonged to clade 2.2. However, two related but distinct genetic subgroups were identified, indicating that two different viruses were circulating in France at the same time and in the same area. Viruses of one subgroup were highly similar to one identified in Bavaria in Germany (A/mallard/Bavaria/1/2006). More surprisingly, French viruses belonging to the other subgroup retained the cleavage motif PQGERKRKKR/G, which is unique among the known HPAI H5N1 viruses. Our results confirmed that multiple H5N1 genogroups were present in Western Europe in early 2006.     

Characterization of H5N1 highly pathogenic avian influenza virus strains isolated from migratory waterfowl in Mongolia on the way back from the southern Asia to their northern territory

H5N1 highly pathogenic avian influenza (HPAI) viruses were isolated from dead wild waterfowl at Khunt, Erkhel, Doityn Tsagaan, Doroo, and Ganga Lakes in Mongolia in July 2005, May 2006, May 2009, July 2009, and May 2010, respectively. The isolates in 2005 and 2006 were classified into genetic clade 2.2, and those in 2009 and 2010 into clade 2.3.2. A/whooper swan/Mongolia/6/2009 (H5N1) experimentally infected ducks and replicated systemically with higher mortality than that of the isolates in 2005 and 2006. Intensive surveillance of avian influenza in migratory waterfowl flying from their nesting lakes in Siberia to Mongolia in every autumn indicate that HPAI viruses have not perpetuated at their nesting lakes until 2009. The present results demonstrate that wild waterfowl were sporadically infected with H5N1 HPAI viruses prevailing in domestic poultry in the southern Asia and died in Mongolia on the way back to their northern territory in spring.     

Evolutionary genetics of highly pathogenic H5N1 avian influenza viruses isolated from whooper swans in northern Japan in 2008

In April and May 2008, highly pathogenic avian influenza viruses subtype H5N1 were isolated from dead or moribund whooper swans in Aomori, Akita and Hokkaido prefectures in northern Japan. To trace the genetic lineage of the isolates, the nucleotide sequences of all eight genes were determined and phylogenetically analyzed. The Japanese strains were nearly identical to chicken viruses isolated in Russia in April 2008 and closely related to viruses isolated from dead wild birds in Hong Kong in 2007–2008. Their HA genes clustered in clade 2.3.2. On the other hand, NA and the other internal genes were closely related to those of clade 2.3.4 viruses (genotype V) whose NP genes originated from an HA clade 2.3.2 virus. In conclusion, the H5N1 viruses isolated in Japan, Russia and Hong Kong were derived from a common ancestor virus belonging to genotype V that was generated from genetic reassortment events between viruses of HA clades 2.3.2 and 2.3.4.     

Characterization of the amantadine-resistant H5N1 highly pathogenic avian influenza variants isolated from quails in Southern China

Highly pathogenic H5N1 avian influenza viruses have spread in poultry and wild birds in Asia, Europe, and Africa since 2003. To evaluate the role of quails in the evolution of influenza A virus, we characterized three H5N1 viruses isolated from quails (QA viruses) in southern China. Phylogenetic analysis indicated that three QA viruses derived from the A/goose/Guangdong/1/96-like lineage and most closely related to HA clade 4 A/chicken/Hong Kong/31.4/02-like viruses. Molecular analysis suggested that QA viruses and clade 4 H5N1 viruses carried consistent residue signatures, such as the characteristic M2 Ser31Asn amantadine-resistance mutation, implying a common origin of these viruses. As revealed by viral pathogenicity tests, these QA viruses could replicate in intranasally infected mice, but were not lethal to them, showing low pathogenicity in mammals. However, they killed all intravenously inoculated chickens, showing high pathogenicity in poultry. Results from amantadine sensitivity tests of wild-type QA viruses and their reverse genetic viruses demonstrated that all QA viruses were resistant to amantadine, and the M2 Ser31Asn mutation was determined as the most likely cause of the increased amantadine-resistance of H5N1 QA viruses. Our study confirmed experimentally that the amino acid at residue 31 in the M2 protein plays a major role in determining the amantadine-resistance phenotype of H5N1 influenza viruses. Our findings provide further evidence that quails may play important roles in the evolution of influenza A viruses, which raises concerns over possible transmissions of H5N1 viruses among poultry, wild birds, and humans.     

Isolation and molecular characterization of the influenza A/H5N1 viruses isolated during the outbreaks of avian influenza among birds in the European part of Russia in 2005: a virus strain with ozeltamivir-resistance mutation was found

The isolation and characterization of the influenza A/H5NI viruses isolated from hens that died during the outbreak of avian influenza in autumn 2005 in the Yandovka village (Tula oblast) and from a wild swan that died near the orifice of the Volga River in the zone of the Karalat Furrow were carried out. Molecular-biologic and phylogenetic analyses were performed with a view of determining possible geographical origin of strains, phylogenetic similarity of viruses and also estimating their pathogenicity, epidemic danger for people, and possible resistance to antiviral drugs. It was shown that the virus belonged to the high pathogenic variants that arose in China as a result of the reassortment of the viruses of the genotypes Z and V that circulated among poultry and wild birds. A number of molecular markers characterizing the high pathogenicity of the virus for gallinaceous birds and mammals were revealed, but the specific mutations in the hemagglutinin gene that promote the high rate of virus replication in a human organism and also the mutations of adaptation to it were not found. It was shown that the variants of the influenza A/H5N1 virus that circulated in this epizootic were sensitive to remantadine. The strain isolated from the wild swan had the mutation causing resistance to Tamiflu/ozeltamivir.     

Interspecies transmission of an H7N3 influenza virus from wild birds to intensively reared domestic poultry in Italy

Since the "bird flu" incident in Hong Kong SAR in 1997, several studies have highlighted the substantial role of domestic birds, such as turkeys and chickens, in the ecology of influenza A viruses. Even if recent evidence suggests that chickens can maintain several influenza serotypes, avian influenza viruses (AIVs) circulating in domestic species are believed to be introduced each time from the wild bird reservoir. However, so far the direct precursor of influenza viruses from domestic birds has never been identified. In this report, we describe the antigenic and genetic characterization of the surface proteins of H7N3 viruses isolated from wild ducks in Italy in 2001 in comparison to H7N3 strains that circulated in Italian turkeys in 2002-2003. The wild and domestic avian strains appeared strictly related at both phenotypic and genetic level: homology percentages in seven of their genes were comprised between 99.8% (for PB2) and 99.1% (for M), and their NA genes differed mainly because of a 23-aminoacid deletion in the NA stalk. Outside this region of the molecule, the NAs of the two virus groups showed 99% similarity. These findings indicate that turkey H7N3 viruses were derived "in toto" from avian influenza strains circulating in wild waterfowl 1 year earlier, and represent an important step towards the comprehension of the mechanisms leading to interspecies transmission and emergence of potentially pandemic influenza viruses.     

The genetic and antigenic diversity of avian influenza viruses isolated from domestic ducks, muscovy ducks, and chickens in northern and southern Vietnam, 2010–2012

To estimate the prevalence of avian influenza virus infection in Vietnam, surveillance was conducted in domestic and wild birds from households, live-bird markets, slaughtering sites, and bird sanctuaries in Vietnam between October 2010 and October 2012. Of the 4,550 samples collected, 226 influenza A virus isolates were obtained from domestic ducks, muscovy ducks, and chickens. Of these, 25 and 22 H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from apparently healthy domestic ducks in live-bird markets and slaughtering sites in northern and southern Vietnam, respectively. The HA genes of H5 viruses isolated from birds in northern Vietnam phylogenetically belonged to the genetic clade 2.3.2.1 and those in southern Vietnam belonged to the genetic clade 1.1. In addition, 39 H3, 12 H4, 1 H5, 93 H6, 2 H7, 18 H9, 3 H10, and 11 H11 viruses were isolated. Phylogenetic and antigenic analyses of the H6 and H9 viruses revealed that they were closely related to the isolates obtained from domestic poultry in China. Phylogenetic analyses of internal gene segments of these isolates revealed that these viruses were circulating in both domestic and wild birds in Asia and reassortment events had occurred frequently. Therefore, it will be important to continue the surveillance and strict controls over the movement and trade of poultry and poultry products in order to eradicate H5N1 HPAIV from Asia.     

Molecular characterization of the glycoprotein genes of H5N1 influenza A viruses isolated in Israel and the Gaza Strip during 2006 outbreaks

Highly pathogenic H5N1 avian influenza A viruses (AIV) have caused outbreaks among domestic poultry and wild aquatic birds in many Asian, European, and African countries since 1997. In March 2006 an avian H5N1 influenza A virus was isolated from poultry in Israel. In the present study we molecularly characterized the hemagglutinin (HA) and neuraminidase (NA) genes of eleven H5N1 viruses isolated from domestic poultry in Israel and Gaza in March–April 2006. Phylogenetic analysis of the HA and NA genes showed that the Israeli and Gazian viruses were closely related to viruses isolated in Egypt in 2006.     

Pathogenicity of H5 influenza viruses for ducks

Summary. Four H5N1 highly pathogenic avian influenza (HPAI) viruses and an avirulent reassortant H5N1 virus were tested for their pathogenicity in domestic ducks. A/chicken/Yamaguchi/7/04 (H5N1) (Ck/Yamaguchi/04) isolated from a dead bird during the HPAI outbreak in Japan and A/duck/Yokohama/aq-10/03 (H5N1) (Dk/Yokohama/03) isolated from duck meat at a quarantine inspection for importation from China replicated in multiple organs including the brain of ducks. The ducks infected with Ck/Yamaguchi/04 did not show any clinical signs, while those infected with Dk/Yokohama/03 showed neurological signs. The ducks infected either with A/Hong Kong/483/97 (H5N1) or A/tern/South Africa/61 (H5N3), or with an avirulent H5N1 reassortant, did not show any clinical signs. Virus-specific antibodies were detected in the sera of the ducks infected with each of the five strains tested, indicating that all of the viral strains infected and replicated in the birds. Dk/Yokohama/03 grew in multiple organs more rapidly than did Ck/Yamaguchi/04. Considerable titers of virus were detected in the brain of the ducks infected with Dk/Yokohama/03 and these birds showed neurological signs. The present results demonstrate that the pathogenicity of influenza viruses for ducks does not correlate with that for chickens and that replication of the virus in the brain is critical for ducks to show neurological signs.     

Investigating a crow die-off in January–February 2011 during the introduction of a new clade of highly pathogenic avian influenza virus H5N1 into Bangladesh

We investigated unusual crow mortality in Bangladesh during January-February 2011 at two sites. Crows of two species, Corvus splendens and C. macrorhynchos, were found sick and dead during the outbreaks. In selected crow roosts, morbidity was ~1 % and mortality was ~4 % during the investigation. Highly pathogenic avian influenza virus H5N1 clade 2.3.2.1 was isolated from dead crows. All isolates were closely related to A/duck/India/02CA10/2011 (H5N1) with 99.8 % and A/crow/Bangladesh/11rs1984-15/2011 (H5N1) virus with 99 % nucleotide sequence identity in their HA genes. The phylogenetic cluster of Bangladesh viruses suggested a common ancestor with viruses found in poultry from India, Myanmar and Nepal. Histopathological changes and immunohistochemistry staining in brain, pancreas, liver, heart, kidney, bursa of Fabricius, rectum, and cloaca were consistent with influenza virus infection. Through our limited investigation in domesticated birds near the crow roosts, we did not identify any samples that tested positive for influenza virus A/H5N1. However, environmental samples collected from live-bird markets near an outbreak site during the month of the outbreaks tested very weakly positive for influenza virus A/H5N1 in clade 2.3.2.1-specific rRT-PCR. Continuation of surveillance in wild and domestic birds may identify evolution of new avian influenza virus and associated public-health risks.     

Evidence of persistence and multiple genetic modifications of H7N7 low-pathogenic avian influenza virus in wild mallards in Poland provided by phylogenetic studies

Genetic characterization of the whole genome of four avian influenza H7N7 viruses isolated in three successive winter seasons (2007 to 2009) from wild mallards in three cities in Poland was performed. All of the tested strains were of low pathogenicity and no molecular marker associated with an increased adaptation to poultry, mammals or resistance to antiviral drugs was found. The major outcome of the phylogenetic studies was that the isolate A/mallard/Poland/446/09 (detected in December 2009) shared a recent common ancestor with A/mallard/Poland/41/09 (isolated in February 2009) in relation to HA and PB1 genes, with A/mallard/Poland/16/09 (found in January 2009) regarding NA and NS genes, and with A/mallard/Poland/01/08 (recovered in December 2007) as regards the NS gene. Interestingly, A/mallard/Poland/16/09 and A/mallard/Poland/446/09 were isolated at the same sampling site almost exactly 1 year apart, which points to resident population of mallards (and other resident waterfowl) as responsible for the perpetuation of avian influenza virus (AIV) in the given area between successive winters. On the other hand, the ornithological data discussed in detail in the paper strongly suggest that the virus transmission between close sites but located in different urban areas is most probably achieved by migratory birds, a fact additionally supported by a close relatedness between different gene segments of Polish H7N7 and AIV detected in wild birds in Europe. A high heterogeneity of the gene pool found in the study is indicative of frequent reassortment events. Additionally, two H7N7 isolates were shown to possess selected genes closely related to AIV detected in domestic poultry in Italy and the Czech Republic. The present study corroborates the importance of active surveillance in wild birds as a valuable tool for early warning of avian influenza in poultry.     

The pathogenesis of influenza in humans

The rapid evolution of influenza A and B viruses contributes to annual influenza epidemics in humans. In addition, pandemics of influenza are also caused by influenza A viruses, whereas influenza B does not have the potential to cause pandemics because there is no animal reservoir of the virus. Study of the genetic differences between influenza A and influenza B viruses, which are restricted to humans, may be informative in understanding the factors that govern mammalian adaptation of influenza A viruses. Aquatic birds provide the natural reservoir for influenza A viruses, but in general, avian influenza is asymptomatic in feral birds. Occasionally, however, highly pathogenic strains of influenza cause serious systemic infections in domestic poultry. The pathogenicity of these strains is related to the presence of a polybasic cleavage sequence in the precursor of the surface glycoprotein haemagglutinin, which makes the glycoprotein susceptible to activation by ubiquitous proteases such as furin and PC6. However, the mechanism of pathogenicity may differ in highly pathogenic strains of human influenza, such as the H1N1 pandemic strain of 1918 and the H5N1 strain involved in the outbreak in Hong Kong in 1997. Binding of host proteases by the viral neuraminidase to assist activation of the haemagglutinin, shortening of the neuraminidase and substitutions in the polymerase gene, PB2, have all been suggested as alternative molecular correlates of pathogenicity of human influenza viruses. Additionally, systemic spread in humans of pathogenic subtypes has not been demonstrated and host factors such as interferons may be crucial in preventing the spread of the virus outside the respiratory tract.