A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Evaluation of Replication and Cross-Reactive Antibody Responses of H2 Subtype Influenza Viruses in Mice and Ferrets

H2 influenza viruses have not circulated in humans since 1968, and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The development of an H2 pandemic influenza virus vaccine candidate should therefore be considered a priority in pandemic influenza preparedness planning. We selected a group of geographically and temporally diverse wild-type H2 influenza viruses and evaluated the kinetics of replication and compared the ability of these viruses to induce a broadly cross-reactive antibody response in mice and ferrets. In both mice and ferrets, A/Japan/305/1957 (H2N2), A/mallard/NY/1978 (H2N2), and A/swine/MO/2006 (H2N3) elicited the broadest cross-reactive antibody responses against heterologous H2 influenza viruses as measured by hemagglutination inhibition and microneutralization assays. These data suggested that these three viruses may be suitable candidates for development as live attenuated H2 pandemic influenza virus vaccines.Influenza pandemics occur when a novel influenza virus enters a population with little preexisting immunity (36). During the pandemics of the last century, novel influenza viruses were introduced either directly from an avian reservoir (34) or were the result of reassortment between contemporaneously circulating human, avian, and swine influenza viruses (5, 29, 36). Due to the lack of preexisting immunity to the novel virus, morbidity and mortality rates are typically higher than in epidemics caused by seasonal influenza viruses (4).Although pandemic preparedness planning has largely focused on the highly pathogenic H5 and H7 avian influenza virus subtypes, the recent emergence of the 2009 pandemic H1N1 viruses underscores the need to consider other influenza virus subtypes as well. Of the 16 hemagglutinin (HA) influenza A virus subtypes that have been identified to date, H1, H2, and H3 have been known to cause influenza pandemics (7, 27), suggesting that these viruses are capable of sustained transmission and can cause disease in humans. While the H1 and H3 subtypes have cocirculated in humans since 1977, H2 influenza viruses have not circulated in humans since 1968 (36) and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The 1957 H2 pandemic virus was a reassortant that derived the HA, neuraminidase (NA), and PB1 genes from an avian virus and the remaining gene segments from the circulating H1N1 virus (15, 30). As H2 subtype viruses continue to circulate in avian reservoirs worldwide (12, 17, 18, 22, 33), they remain a potential pandemic threat. The development of an H2 influenza virus vaccine candidate should therefore be considered a priority in future pandemic influenza preparedness planning.Given the low likelihood that a previously selected vaccine virus will exactly match the pandemic virus, the ability to elicit a broadly cross-reactive antibody response to antigenically distinct viruses within a subtype is an important consideration in the selection of a pandemic influenza vaccine candidate. Previous studies have examined the ability of inactivated H2 influenza viruses to provide cross-protection against mouse-adapted variants of reassortant human viruses and an avian H2 influenza virus from 1978 (9, 14). Given the potential for live attenuated influenza virus vaccines to confer a great breadth of heterologous cross-protection (1, 2, 6, 35), we recently conducted a study evaluating cold-adapted A/Ann Arbor/6/1960 (AA CA), an H2 influenza virus used as the backbone of the seasonal live attenuated influenza A virus vaccine currently licensed in the United States (3). However, as H2 influenza virus continues to circulate widely and appear in migratory birds (10, 24, 26), in poultry markets (20), and in swine (21), with evidence of interregional gene transmission (19, 22), a more extensive evaluation of recent isolates may be warranted in the selection of a potential H2 pandemic vaccine candidate.H2 influenza viruses fall into three main lineages: a human lineage, a North American avian lineage, and a Eurasian avian lineage (29). In addition to viruses whose replicative ability in mammals has previously been established (11, 21, 23, 25), we selected a group of geographically and temporally diverse H2 influenza viruses from each lineage. We evaluated the kinetics of replication of each of these viruses in mice and ferrets and compared the abilities of these viruses to induce a broadly cross-reactive antibody response to determine which of these viruses would be suitable for further development as an H2 pandemic influenza vaccine candidate.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).     

The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model

Adaptation of influenza A viruses to a new host species usually involves the mutation of one or more of the eight viral gene segments, and the molecular basis for host range restriction is still poorly understood. To investigate the molecular changes that occur during adaptation of a low-pathogenic avian influenza virus subtype commonly isolated from migratory birds to a mammalian host, we serially passaged the avirulent wild-bird H5N2 strain A/Aquatic bird/Korea/W81/05 (W81) in the lungs of mice. The resulting mouse-adapted strain (ma81) was highly virulent (50% mouse lethal dose = 2.6 log₁₀ 50% tissue culture infective dose) and highly lethal. Nonconserved mutations were observed in six viral genes (those for PB2, PB1, PA, HA, NA, and M). Reverse genetic experiments substituting viral genes and mutations demonstrated that the PA gene was a determinant of the enhanced virulence in mice and that a Thr-to-Iso substitution at position 97 of PA played a key role. In growth kinetics studies, ma81 showed enhanced replication in mammalian but not avian cell lines; the PA_97I mutation in strain W81 increased its replicative fitness in mice but not in chickens. The high virulence associated with the PA_97I mutation in mice corresponded to considerably enhanced polymerase activity in mammalian cells. Furthermore, this characteristic mutation is not conserved among avian influenza viruses but is prevalent among mouse-adapted strains, indicating a host-dependent mutation. To our knowledge, this is the first study that the isoleucine residue at position 97 in PA plays a key role in enhanced virulence in mice and is implicated in the adaptation of avian influenza viruses to mammalian hosts.Migratory waterfowl are the natural reservoir of influenza A viruses (11, 53). The viruses replicate efficiently in their natural hosts but replicate poorly if at all in other species (53). However, these viruses can undergo adaptation or genetic reassortment to infect other hosts (43, 44, 53), including humans. Since 1997, the World Health Organization has documented more than 400 laboratory-confirmed cases of human infection with H5N1 avian influenza virus (54).The molecular basis of influenza virus host range restriction and adaptation to a new host species is poorly understood. Mutations associated with cross-species adaptation are thought to be associated with increased virulence (30). Therefore, studies in animal models have attempted to identify the viral molecular determinants of virulence in specific hosts. Reverse genetics (Rg) methods have also identified genetic differences that affect virus virulence and host range, including changes in the viral internal proteins. Experimental infection of mouse lungs is an effective approach for understanding influenza virus virulence and adaptation (reviewed by A. C. Ward [51]). To acquire virulence in mice, influenza A viruses usually must adapt to these hosts over several consecutive generations (serial passages) in the lungs or brain (1, 25, 30). Previous studies have found that the acquisition of virulence during adaptation in the mouse model is associated with mutations in the HA, NP, NA, M, and NS genes and one or more polymerase genes (2, 3, 18, 36, 42, 51). The polymerase basic protein 2 (PB2) gene is a particularly well-characterized polymerase subunit (7, 23, 40, 46). The PB1 and polymerase acidic protein (PA) genes have been implicated in mouse lung virulence (5, 18, 36, 39, 49) but have shown no evidence of having acquired mutations during adaptation (52). However, the many studies conducted to date have focused mainly on highly pathogenic avian influenza (HPAI) viruses such as the H1N1, H5N1, and H7N7 subtypes (7, 23, 48, 50).Various low-pathogenic avian influenza (LPAI) viruses are considered to be potential genetic contributors to the next pandemic strain. Lee et al. (2009) recently reported the presence of avian-like LPAI H5N2 viruses in a number of Korean swine and proposed that the efficient transmissibility of the swine-adapted H5N2 virus could facilitate spread of the virus. They suggested that this adapted virus could potentially serve as a model for pandemic outbreaks of HPAI (e.g., H5N1 and H7N7) virus or could become a pandemic strain itself (21). These findings prompted our interest in the adaptation of an LPAI virus often harbored by wild migratory birds of South Korea. In our ongoing surveillance from 2004 to 2008, approximately 27% of the viruses isolated were of the H5N2 subtype (unpublished data). Studies show that influenza viruses with different genetic backgrounds can acquire different mutations during adaptation in mice. Therefore, we sought to determine whether this common H5N2 virus (nonlethal in mice) would undergo changes different from those observed in highly virulent viruses during adaptation in mice. Wild-bird influenza virus strain A/Aquatic bird/Korea/W81/05 (W81) was adapted in mice over 11 passages and became highly virulent. To identify molecular determinants of this adaptation and altered virulence, we used Rg-generated recombinant viruses to compare the parental and mouse-adapted strains. Here we show that the PA subunit of the polymerase complex, independently of PB2, contributed to adaptation and increased virulence in our mammalian model.     

Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused devastating outbreaks in avian species during the past decade. After emerging in the Guangdong province of China in 1996, H5N1 viruses have extended their geographic distribution from Asia into Europe and Africa (45, 51). Sporadic transmission of H5N1 viruses from infected birds to humans has resulted in over 380 laboratory-confirmed infections and a case fatality rate of ∼60% since 2003 (48). Currently circulating H5N1 viruses lack the ability to undergo efficient and sustained transmission among humans although instances of limited human-to-human transmission have been reported (13, 41). If H5N1 viruses were to acquire genetic changes that confer efficient transmissibility among humans, then another pandemic would likely occur.The pandemics of 1957 and 1968 highlight the importance of genetic reassortment between avian and human influenza viruses as a mechanism for the generation of human pandemic strains (15, 46, 47). The structural separation of the influenza virus genome into eight independent genes allows formation of hybrid progeny viruses during coinfections. The 1957 H2N2 and 1968 H3N2 pandemic viruses acquired the hemagglutinin (HA) and PB1 genes, with or without the neuraminidase (NA) gene, respectively, from an avian virus progenitor (14, 33). The remaining genes of these pandemic reassortants were derived from a contemporary human virus (14, 33). The host species in which such human pandemic strains were generated by reassortment between human and avian viruses is not known. However, coinfection of the same cell with both human and avian viruses must have occurred, even though human and avian influenza viruses have preferences for different sialic acid receptor structures present on cell surface glycoproteins and glycolipids (20, 30). The HA of human viruses preferentially binds α(2,6)-linked sialic acids while that of avian viruses preferentially bind α(2,3)-linked sialic acids (3, 12). Cells possessing both of these receptors could support coinfection of avian and human viruses, leading to reassortment.Human respiratory tract epithelial cells can possess surface glycans with α(2,3)- and α(2,6)-linked sialic acids and as such represent a potential host for the generation of avian-human reassortant viruses (24, 35). The general distribution of surface α(2,3)- and α(2,6)-linked sialic acids varies among cells of the human upper and lower respiratory tracts, which are anatomically separated by the larynx. Recent studies have shown that α(2,3)-linked sialic acids are present in tissues of the human lower respiratory tract (i.e., lung alveolar cells) (24, 35) as well as tissues of the human upper respiratory tract (24). Consistent with these findings, HPAI H5N1 viruses have been shown to attach to and infect tissues belonging to the lower respiratory tract (i.e., trachea, bronchi, and lung) (5, 25, 35, 40, 42, 43) as well as tissues belonging to the upper respiratory tract (i.e., nasopharyngeal, adenoid, and tonsillar) (25). Glycans with α(2,6)-linked sialic acids are more widespread on epithelial cells of the upper airways than lung alveoli (24, 35). In accordance, human seasonal influenza viruses preferentially attach to and infect cells of the upper respiratory tract (6, 25, 35, 43). If cells with both types of receptors are present in the human respiratory tract, simultaneous infection of a person with both human and avian viruses could generate reassortant viruses.Although viruses derived by reassortment between avian H5N1 and human H3N2 progenitors have been generated in vitro (17), reassortment between these avian and human strains in a coinfected mammalian host has not been shown. Furthermore, our knowledge of the genetic and phenotypic repertoire of such reassortants generated in vivo and their potential for transmission to uninfected hosts is limited (2, 17). In the present study, we used the ferret model to better understand the generation of reassortant viruses in a host coinfected with contemporary avian (H5N1) and human (H3N2) viruses and the extent to which such reassortants replicate and transmit from animal to animal. The domestic ferret (Mustela putoris) serves as an ideal small-animal model for influenza because ferrets are susceptible to human and avian influenza viruses, including HPAI H5N1 viruses, and reflect the relative transmissibility of human and avian influenza viruses in humans (9, 17, 18, 31, 36, 39, 53). Our study revealed that coinfection of ferrets reproducibly generated reassortant viruses that could be recovered from tissues within and extending toward the upper respiratory tract. Although H5 reassortant viruses were recovered from the upper airways, they displayed no transmissibility to contact ferrets, suggesting that additional functional changes are required for these viral subtypes to become pandemic within human populations.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

A Single Immunization with CoVaccine HT-Adjuvanted H5N1 Influenza Virus Vaccine Induces Protective Cellular and Humoral Immune Responses in Ferrets

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 μg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.Since the first human case of infection with a highly pathogenic avian influenza A virus of the H5N1 subtype in 1997 (9, 10, 37), hundreds of zoonotic transmissions have been reported, with a high case-fatality rate (10, 44). Since these viruses continue to circulate among domestic birds and human cases are regularly reported, it is feared that they will adapt to their new host or exchange gene segments with other influenza A viruses, become transmissible from human to human, and cause a new pandemic. Recently, a novel influenza A virus of the H1N1 subtype emerged. This virus, which originated from pigs, was transmitted between humans efficiently, resulting in the first influenza pandemic of the 21st century (8, 45). Although millions of people have been inoculated with the (H1N1)2009 virus, the case-fatality rate was relatively low compared to that for infections with the H5N1 viruses (11, 31). However, the unexpected pandemic caused by influenza A/H1N1(2009) viruses has further highlighted the importance of rapid availability of safe and effective pandemic influenza virus vaccines. Other key issues for the development of pandemic influenza A virus vaccines include optimal use of the existing (limited) capacity for production of viral antigen and effectiveness against viruses that are antigenically distinct. Ideally, a single administration of a low dose of antigen would be sufficient to induce protective immunity against the homologous strain and heterologous antigenic variant strains. However, since the population at large will be immunologically naïve to a newly introduced virus, high doses of antigen are required to induce protective immunity in unprimed subjects (23, 36). The use of safe and effective adjuvants in pandemic influenza virus vaccines is considered a dose-sparing strategy. Clinical trials evaluating candidate inactivated influenza A/H5N1 virus vaccines showed that the use of adjuvants can increase their immunogenicity and broaden the specificity of the induced antibody responses (2, 7, 19, 23, 27, 36, 41). These research efforts have resulted in the licensing of adjuvanted vaccines against seasonal and pandemic influenza viruses (17). The protective efficacy of immune responses induced with candidate influenza A/H5N1 virus vaccines was demonstrated in ferrets after two immunizations (1, 22, 24, 25) or after a single immunization. The latter was achieved with a low dose of antigen in combination with the adjuvant Iscomatrix (26).Recently, a novel adjuvant that consists of a sucrose fatty acid sulfate ester (SFASE) immobilized on the oil droplets of a submicrometer emulsion of squalane in water has been developed (4). It has been demonstrated that the addition of this novel adjuvant, called CoVaccine HT, to multiple antigens increased the immune response to these antigens in pigs and horses and was well tolerated in both species (4, 16, 40). Furthermore, it was shown that the use of CoVaccine HT increased the virus-specific antibody responses in mice and ferrets after vaccination with a cell culture-derived whole inactivated influenza A/H5N1 virus vaccine (5, 13). One of the mode of actions of CoVaccine HT is the activation of antigen-presenting cells such as dendritic cells, most likely through Toll-like receptor 4 (TLR4) signaling (5).In the present study, we evaluated the protective potential of CoVaccine HT-adjuvanted cell culture-derived whole inactivated influenza A/H5N1 virus (WIV) vaccine in the ferret model, which is considered the most suitable animal model for the evaluation of candidate influenza virus vaccines (6, 14, 15). To this end, ferrets were vaccinated once or twice with various antigen doses with or without the adjuvant to test whether dose sparing could be achieved. The use of CoVaccine HT increased virus-specific antibody responses and T cell responses. A single administration of 3.8 μg hemagglutinin (HA) of WIV NIBRG-14 vaccine preparation in combination with CoVaccine HT conferred protection against challenge infection with the homologous highly pathogenic A/H5N1 virus strain A/VN/1194/04 and partial protection against infection with a heterologous, antigenically distinct strain, A/IND/5/05. Therefore, it was concluded that the use of CoVaccine HT in inactivated influenza virus vaccines induced protective virus-specific humoral and cell-mediated immune responses and that it could be suitable as adjuvant in (pre)pandemic A/H5N1 virus vaccines. Further clinical testing of these candidate vaccines seems to be warranted.     

Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.Since the first detection of the Asian lineage of highly pathogenic avian influenza (HPAI) virus (H5N1) in southern China in 1996, H5N1 virus infection in birds has continued for 13 years in Asia, acquiring pathogenicity not only in birds but also in mammals. In 1997, the H5N1 Hong Kong isolates caused illness and death in a variety of terrestrial birds and even in humans (9, 37, 48, 49). In 2001, emerging H5N1 Hong Kong isolates were more pathogenic to chickens and the mean death time (MDT) was 2 days without any prior clinical signs (12). In 2003 to 2004, the H5N1 epizootic occurred simultaneously in East Asian countries (22, 30). The 2003/2004 H5N1 isolates caused death in taxonomically diverse avian species, including domestic ducks (46, 47, 51), and humans (7, 55). Furthermore, the first indication of wild aquatic bird involvement occurred at recreational parks in Hong Kong in late 2002 to 2003 (46), and then migratory aquatic bird die-off occurred in 2005 at Qinghai Lake in China (6, 24). The broad host spectrum and increased pathogenicity of H5N1 viruses to diverse bird species raise serious concerns about the worldwide spread of the virus by migratory birds.According to the international criteria, HPAI viruses are defined by over 75% mortality in 4- to 8-week-old chickens following an intravenous pathogenicity test or an intravenous pathogenicity index (IVPI) of more than 1.2 in 6-week-old chickens (34); however, there are some variations in pathogenicity intensity among the HPAI viruses in chickens (1, 3, 5, 12, 15, 28, 31, 48, 50-52, 57). Most of the HPAI viruses that were isolated before 1996 cause severe clinical signs (e.g., ruffled feathers, depression, labored breathing, and neurological signs) and severe gross lesions (e.g., head and face edema, cyanosis, subcutaneous hemorrhages in combs and leg shanks, and necrosis of combs and wattles) in chickens (1, 3, 15, 31, 50, 52, 57). These viruses usually kill chickens 3 to 6 days after intranasal inoculation. On the other hand, the recently emerged Asian H5N1 HPAI viruses are more virulent and kill chickens within 1 to 2 days without causing typical clinical signs and gross lesions (5, 12, 27, 33, 48, 51), although some Asian H5N1 viruses, such as A/Goose/Guangdong/2/96 (23), A/goose/Hong Kong/437-10/99 (17), and A/chicken/Indonesia/7/03 (58), were less virulent. To successfully control HPAI in poultry, it is important to better understand the mechanisms of increased pathogenicity of recent H5N1 HPAI viruses in chickens.The Asian H5N1 HPAI virus has another important characteristic, which is its capability of crossing host-species barriers. It was reported that the H5N1 virus can infect and cause death in mammals such as mice (5, 9, 12, 14, 29), cats (21), tigers (2), ferrets (11, 26), monkeys (40), and humans (7, 49, 55). High-level inductions of proinflammatory cytokines in mammals infected with the H5N1 viruses, referred to as “cytokine storms,” have been hypothesized to contribute to the severity of pathological changes and ultimate death (4, 7, 13, 45, 55). Cytokine and chemokine dysregulation was detected in clinical cases of H5N1-infected humans (8, 13, 36) and also in monkeys experimentally infected with the H1N1 Spanish flu strain (20). In a mouse model, lymphocyte apoptosis and cytokine dysregulation have been proposed to contribute to the severity of the disease caused by H5N1 (56). Investigations with transgenic mice deficient in each cytokine gene suggest that tumor necrosis factor alpha (TNF-α) may contribute to morbidity and interleukin-1 (IL-1) may be important for virus clearance (53). However, mice deficient in TNF-α or IL-6 succumb to infection with H5N1, and cytokine inhibition treatment does not prevent death (42), suggesting that therapies targeting the virus rather than cytokines may be preferable. Thus, the significance of elevated proinflammatory cytokine responses in the pathogenesis of H5N1-infected mammals requires further studies.In contrast, little is known about proinflammatory cytokine responses and their roles in pathogenicity in chickens infected with HPAI viruses, including the recent Asian H5N1 viruses. It was reported that infection with an HPAI virus results in upregulation of gene expression of gamma interferon (IFN-γ) and inducible nitric oxide synthase (58). However, the roles of proinflammatory cytokines in disease severity and outcomes in chickens infected systemically with HPAI viruses are largely unknown. The less-virulent Asian H5N1 virus, which causes severe clinical signs and gross lesions in chickens (17, 23, 27, 58), would be a valuable tool for investigating the role of proinflammatory cytokines in chickens infected with HPAI viruses, as well as for exploring the pathogenesis of the more-virulent Asian H5N1 HPAI virus, because of the antigenic and molecular similarities between them.In this study, we compared the pathogenicities in chickens of the less-virulent and more-virulent Asian H5N1 HPAI viruses based on MDT, fever, cytokine responses, and viral replication. Our results suggest that the shift in the Asian H5N1 virus to increased virulence may be associated with efficient and rapid replication of the virus in chickens, accompanied by early destruction of host immune responses and followed by peracute death before fever can be induced. Finally, we discuss candidate genes that may account for the high pathogenicity of Asian H5N1 HPAI viruses in chickens.     

Molecular Analysis of H7 Avian Influenza Viruses from Australia and New Zealand: Genetic Diversity and Relationships from 1976 to 2007

Full-genome sequencing of 11 Australian and 1 New Zealand avian influenza A virus isolate (all subtype H7) has enabled comparison of the sequences of each of the genome segments to those of other subtype H7 avian influenza A viruses. The inference of phylogenetic relationships for each segment has been used to develop a model of the natural history of these viruses in Australia. Phylogenetic analysis of the hemagglutinin segment indicates that the Australian H7 isolates form a monophyletic clade. This pattern is consistent with the long-term, independent evolution that is, in this instance, associated with geographic regions. On the basis of the analysis of the other H7 hemagglutinin sequences, three other geographic regions for which similar monophyletic clades have been observed were confirmed. These regions are Eurasia plus Africa, North America, and South America. Analysis of the neuraminidase sequences from the H7N1, H7N3, and H7N7 genomes revealed the same region-based relationships. This pattern of independent evolution of Australian isolates is supported by the results of analysis of each of the six remaining genomic segments. These results, in conjunction with the occurrence of five different combinations of neuraminidase subtypes (H7N2, H7N3, H7N4, H7N6, H7N7) among the 11 Australian isolates, suggest that the maintenance host(s) is nearly exclusively associated with Australia. The single lineage of Australian H7 hemagglutinin sequences, despite the occurrence of multiple neuraminidase types, suggests the existence of a genetic pool from which a variety of reassortants arise rather than the presence of a small number of stable viral clones. This pattern of evolution is likely to occur in each of the regions mentioned above.The emergence of highly pathogenic avian influenza viruses of subtype H5N1 as a potential human pandemic disease threat has focused attention on the roles that wild birds play in the maintenance and distribution of avian influenza viruses (18, 22). Moreover, the H5 and H7 subtypes of avian influenza A virus are major causes of economic loss in poultry production through disease. In Australia, there have been five documented outbreaks of H7 subtype avian influenza A virus disease, with evidence of adaptation to the poultry host being provided by sequence data supporting the presence of high-pathogenicity avian influenza virus (HPAI) isolates in poultry. Waterfowl (Anseriformes order, particularly ducks, geese, and swans) and the waders and gulls (Charadriiformes order, particularly gulls, terns, and waders) have been found to be the major global natural reservoirs of influenza A viruses. Transmission of avian influenza viruses from wild birds to production poultry and geographic spread are dependent upon the migratory behavior of the wild bird reservoir hosts. Members of the Anseriformes and Charadriiformes orders undertake both irregular and regular transcontinental and intercontinental migrations. During these migrations, large numbers of birds congregate at aquatic feeding locations, providing ideal sites for cross-species transmission of avian influenza viruses. A variety of mechanisms have been observed whereby influenza A viruses adapt rapidly. These include genetic shifts facilitated through genome segment reassortment, as well as genetic drift through the insertion, deletion, and substitution of nucleotides. The error-prone RNA replication and a lack of error correction are the causes of drift. In vivo, this results in viral genetic diversity within any viral sample, or a quasispecies, thus providing a pool of closely related variant viruses from enabling events, such as viral adaptation to new hosts (25). Long-term sampling of water birds in North America and Europe has started to elucidate the ecology and biology of the avian influenza A virus types in the natural reservoirs in these regions (8, 18, 22). There is a suggestion that two superfamilies, the Eurasia (which in the context of this paper includes Europe, Asia, and Africa) and the Americas superfamilies, exist; however, the extent of overlap and the rate of transfer of influenza viruses between these two regions are not well-defined. Recent studies suggest that intercontinental virus exchange is slow and limited (17), while a detailed analysis of the differences between H7 hemagglutinin (HA) segments circulating in Europe and China showed that the H7 hemagglutinin segments shared a recent common ancestor and limited sequence divergence on a background of multiple reassortant virus genotypes between 1999 and 2005 (7).Avian influenza A viruses of the Oceania region (Australia, New Zealand, and southwest Pacific) have been far less well studied (3). Australia and New Zealand are at the southern extremity of a number of major bird migration pathways. Waders in the Charadriidae family migrate to south and southeastern Australia and New Zealand from their summer breeding grounds in Arctic regions of Siberia and Alaska, where they freely mix with the same or other species which migrate into the shared breeding grounds of Eurasia and the Americas (30). Pelagic seabirds of the Procellariformes order breed on and around Australian and New Zealand coasts during the southern hemisphere summer and migrate to maritime regions of the northern Pacific associated with Japan, Russia, and Alaska. Some move as far as the west coasts of North and South America (28). Unlike North and South America and Europe, where regular migrations of ducks, geese, swans, etc., are established, the members of the Anatidae family (ducks, etc.) in Australia and New Zealand are mainly endemic residents (30). However, within Australia, ducks undertake long-distance movements in response to water availability. Movements of waterfowl from northern Australia to nearby areas of Southeast Asia are believed to occur but are limited, as suggested by Wallace''s Line (19). Generally, these waterfowl movements have not been well studied (30). The risks to Australian poultry production systems by movement of H5N1 via migratory shorebirds and nomadic wildfowl have been assessed to be low using risk-based analysis techniques (9, 10).Regular and extensive surveillance sampling of migratory birds has been undertaken in North America and northern Europe (17, 18). The findings have shed significant insights into the ecology of the viruses and their hosts (8, 17). In contrast, surveillance sampling of wild birds in Asia and Oceania has been spasmodic and sparse, until the recent emergence of H5N1 highly pathogenic avian influenza virus as a poultry and human disease threat. Spasmodic and small-scale outbreaks of highly pathogenic avian influenza virus have occurred in Australian poultry production flocks located in the southeastern region of the continent. These poultry production areas are concentrated close to large human population centers (26, 33, 34). Each of the Australian outbreaks has been rapidly controlled by slaughter of infected flocks. All have been caused by avian influenza viruses of the H7 subtype, which appear to have entered production poultry from water birds, possibly wild ducks, via contaminated water supplies used on the poultry farms. Disease has occurred on five occasions: 1976 (H7N7), 1985 (H7N7), 1992 (H7N3), 1994 (H7N3), and 1997 (H7N4) (13, 14, 26, 27, 31, 34). National on-farm biosecurity measures have been focused on reducing the likelihood of future outbreaks. The availability of avian influenza virus isolates from poultry and wild birds associated with these outbreaks, along with a small number of subtype H7 avian influenza viruses isolated from wild ducks during recent national surveillance programs in Australia and New Zealand, provided the opportunity to explore the relationships of Australian and New Zealand subtype H7 avian influenza virus isolates with viruses circulating elsewhere in the world.     

Transmission of Pandemic H1N1 Influenza Virus and Impact of Prior Exposure to Seasonal Strains or Interferon Treatment

Novel swine-origin influenza viruses of the H1N1 subtype were first detected in humans in April 2009. As of 12 August 2009, 180,000 cases had been reported globally. Despite the fact that they are of the same antigenic subtype as seasonal influenza viruses circulating in humans since 1977, these viruses continue to spread and have caused the first influenza pandemic since 1968. Here we show that a pandemic H1N1 strain replicates in and transmits among guinea pigs with similar efficiency to that of a seasonal H3N2 influenza virus. This transmission was, however, partially disrupted when guinea pigs had preexisting immunity to recent human isolates of either the H1N1 or H3N2 subtype and was fully blocked through daily intranasal administration of interferon to either inoculated or exposed animals. Our results suggest that partial immunity resulting from prior exposure to conventional human strains may blunt the impact of pandemic H1N1 viruses in the human population. In addition, the use of interferon as an antiviral prophylaxis may be an effective way to limit spread in at-risk populations.A pandemic of novel swine-origin influenza virus (H1N1) is developing rapidly. As of 12 August 2009, nearly 180,000 cases had been reported to the WHO from around the globe (36). Sustained human-to-human transmission has furthermore been observed in multiple countries, prompting the WHO to declare a public health emergency of international concern and to raise the pandemic alert level to phase 6 (7).Swine are a natural host of influenza viruses, and although sporadic incidences of human infection with swine influenza viruses occur (8, 9, 14, 29, 35), human-to-human transmission is rare. H1N1 influenza viruses have likely circulated in swine since shortly after the 1918 human influenza pandemic (38). From the 1930s, when a swine influenza virus was first isolated, to the late 1990s, this classical swine lineage has remained relatively stable antigenically (34). In the late 1990s, however, genetic reassortment between a human H3N2 virus, a North American avian virus, and a classical swine influenza virus produced a triple reassortant virus, which subsequently spread among North American swine (34). Further reassortment events involving human influenza viruses led to the emergence in pigs of triple reassortants of the H1N1 and H1N2 subtypes (34). None of these swine viruses have demonstrated the potential for sustained human-to-human transmission.The swine-origin influenza viruses now emerging in the human population possess a previously uncharacterized constellation of eight genes (28). The NA and M segments derive from a Eurasian swine influenza virus lineage, having entered pigs from the avian reservoir around 1979, while the HA, NP, and NS segments are of the classical swine lineage and the PA, PB1, and PB2 segments derive from the North American triple reassortant swine lineage (13). This unique combination of genetic elements (segments from multiple swine influenza virus lineages, some of them derived from avian and human influenza viruses) may account for the improved fitness of pandemic H1N1 viruses, relative to that of previous swine isolates, in humans.Several uncertainties remain about how this outbreak will develop over time. Although the novel H1N1 virus has spread over a broad geographical area, the number of people known to be infected remains low in many countries, which could be due, at least in part, to the lack of optimal transmission of influenza viruses outside the winter season; thus, it is unclear at this point whether the new virus will become established in the long term. Two major factors will shape the epidemiology of pandemic H1N1 viruses in the coming months and years: the intrinsic transmissibility of the virus and the degree of protection offered by previous exposure to seasonal human strains. Initial estimates of the reproductive number (R₀) have been made based on the epidemiology of the virus to date and suggest that its rate of spread is intermediate between that of seasonal flu and that of previous pandemic strains (3, 11). However, more precise estimates of R₀ will depend on better surveillance data in the future. The transmission phenotype of pandemic H1N1 viruses in a ferret model was also recently reported and was found to be similar to (16, 27) or less efficient (25) than that of seasonal H1N1 strains. The reason for this discrepancy in the ferret model is unclear.Importantly, in considering the human population, the impact of immunity against seasonal strains on the transmission potential of pandemic H1N1 viruses is not clear. According to conventional wisdom, an influenza virus must be of a hemagglutinin (HA) subtype which is novel to the human population in order to cause a pandemic (18, 38). Analysis of human sera collected from individuals with diverse influenza virus exposure histories has indicated that in those born in the early part of the 20th century, neutralizing activity against A/California/04/09 (Cal/04/09) virus is often present (16). Conversely, serological analyses of ferret postinfection sera (13) and human pre- and postvaccination sera (4a) revealed that neutralizing antibodies against recently circulating human H1N1 viruses do not react with pandemic H1N1 isolates. These serological findings may explain the relatively small number of cases seen to date in individuals greater than 65 years of age (6). Even in the absence of neutralizing antibodies, however, a measure of immune protection sufficient to dampen transmission may be present in a host who has recently experienced seasonal influenza (10). If, on the other hand, transmission is high and immunity is low, then pandemic H1N1 strains will likely continue to spread rapidly through the population. In this situation, a range of pharmaceutical interventions will be needed to dampen the public health impact of the pandemic.Herein we used the guinea pig model (4, 21-24, 26, 30) to assess the transmissibility of the pandemic H1N1 strains Cal/04/09 and A/Netherlands/602/09 (NL/602/09) relative to that of previous human and swine influenza viruses. To better mimic the human situation, we then tested whether the efficiency of transmission is decreased by preexisting immunity to recent human H1N1 or H3N2 influenza viruses. Finally, we assessed the efficacy of intranasal treatment with type I interferon (IFN) in limiting the replication and transmission of pandemic H1N1 viruses.     

Insertion of a Multibasic Cleavage Motif into the Hemagglutinin of a Low-Pathogenic Avian Influenza H6N1 Virus Induces a Highly Pathogenic Phenotype

The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR↓G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA₀ (the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtype-specific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype.Wild birds represent the natural reservoir of avian influenza A viruses in nature (43). Influenza A viruses are classified on the basis of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. In wild birds throughout the world, influenza A viruses representing 16 HA and 9 NA antigenic subtypes have been found in numerous combinations (also called subtypes, e.g., H1N1, H6N1) (12). Besides classification based on the antigenic properties of HA and NA, avian influenza A viruses can also be classified based on their pathogenic phenotype in chickens. Highly pathogenic avian influenza (HPAI) virus, an acute generalized disease of poultry in which mortality may be as high as 100%, is restricted to subtypes H5 and H7. Other avian influenza A virus subtypes are generally low-pathogenic avian influenza (LPAI) viruses that cause much milder, primarily respiratory disease in poultry, sometimes with loss of egg production (6).The HA protein of influenza A virus is initially synthesized as a single polypeptide precursor (HA₀), which is cleaved into HA₁ and HA₂ subunits by host cell proteases. The mature HA protein mediates binding of the virus to host cells, followed by endocytosis and HA-mediated fusion with endosomal membranes (43). Influenza viruses of subtypes H5 and H7 may become highly pathogenic after introduction into poultry and cause outbreaks of HPAI. The switch from an LPAI phenotype to the HPAI phenotype of these H5 and H7 influenza A viruses is achieved by the introduction of basic amino acid residues into the HA₀ cleavage site by substitution or insertion, resulting in the so-called multibasic cleavage site (MBCS), which facilitates systemic virus replication (4, 5, 14, 44). The cleavage of the HA₀ of LPAI viruses is restricted to trypsin-like proteases which recognize the XXX(R/K)↓G cleavage motif, where the downward arrow indicates the site of cleavage. Replication of these LPAI viruses is therefore restricted to sites in the host where these enzymes are expressed, i.e., the respiratory and intestinal tract (32, 38). The introduction of an RX(R/K)R↓G or R(R/K)XR↓G minimal MBCS motif into the H5 and H7 subtype viruses facilitates the recognition and cleavage of the HA₀ by ubiquitous proprotein convertases, such as furin (20, 32, 41, 45). H5 influenza A viruses with a minimal MBCS motif only have the highly pathogenic phenotype if the masking glycosylation site at position 11 in the HA is replaced by a nonglycosylation site. Otherwise, at least one additional basic amino acid has to be inserted to allow the shift from an LPAI virus phenotype to an HPAI virus phenotype to occur (15, 18, 21, 22, 28). No information is available on the minimal prerequisites of H7 influenza A viruses to become highly pathogenic, but all HPAI H7 viruses have at least 2 basic amino acid insertions in the HA₀ cleavage site (22). HA₀ with the MBCS is activated in a broad range of different host cells and therefore enables HPAI viruses to replicate systemically in poultry (46). To date, little is known about the apparent subtype-specific nature of the introduction of the MBCS into LPAI viruses and the evolutionary processes involved in the emergence of HPAI viruses. When an MBCS was introduced in a laboratory-adapted strain of influenza virus, A/Duck/Ukraine/1/1963 (H3N8), it did not result in a dramatic change in pathogenic phenotype (35). Here, the effect of the introduction of an MBCS into a primary LPAI H6N1 virus, A/Mallard/Sweden/81/2002, is described. The introduction of an MBCS resulted in trypsin-independent replication in vitro and enhanced pathogenesis in a chicken model. Understanding the basis of the HA subtype specificity of the introduction of an MBCS into avian influenza viruses will lead to a better understanding of potential molecular restrictions involved in emergence of HPAI outbreaks.     

Competitive Fitness of Oseltamivir-Sensitive and -Resistant Highly Pathogenic H5N1 Influenza Viruses in a Ferret Model

The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host.The neuraminidase (NA) inhibitors (orally administered oseltamivir and inhaled zanamivir) are currently an important class of antiviral drugs available for the treatment of seasonal and pandemic influenza. Although administration of NA inhibitors may significantly reduce influenza virus transmission, it risks the emergence of drug-resistant variants (16, 32). The impact of drug resistance would depend on the fitness (i.e., infectivity in vitro and virulence and transmissibility in vivo) of the resistant virus. If the resistance mutation only modestly reduces the virus'' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. The unexpected natural emergence and spread of oseltamivir-resistant variants (carrying the H274Y NA amino acid substitution) among seasonal H1N1 influenza viruses of the A/Brisbane/59/07 lineage demonstrated that drug-resistant viruses can be highly fit and transmissible in humans (11, 22, 29), although the fitness of these variants is not completely understood. They are hypothesized to have lower NA receptor affinity and more-optimal NA and hemagglutinin (HA) functional balance than do wild-type (WT) viruses (38). Fortunately, oseltamivir-resistant variants have rarely been reported to occur among the novel pandemic H1N1 influenza viruses that emerged in April 2009; therefore, initial data suggest that currently circulating wild-type viruses possibly possess greater fitness than drug-resistant viruses (45), although only retrospective epidemiological data can provide a conclusive answer. The key questions are whether the risk posed by NA inhibitor-resistant viruses can be assessed experimentally and what the most reliable approach may be.All NA inhibitor-resistant influenza viruses characterized to date have contained specific mutations in the NA molecule. Clinically derived drug-resistant viruses have carried mutations that are NA subtype specific and differ in accordance with the NA inhibitor used (12, 35). The most commonly observed mutations are H274Y and N294S in the influenza A N1 NA subtype, E119A/G/D/V and R292K in the N2 NA subtype, and R152K and D198N in influenza B viruses (35, 36). The fitness of NA inhibitor-resistant viruses has been studied in vitro and in vivo. Many groups have assessed their replicative capacity in MDCK cells, but this assay system can yield anomalous results (49), particularly in the case of low-passage clinical isolates. The mismatch between virus specificity and cellular receptors can be overcome by using cell lines engineered to express human-like α-2,6-linked sialyl cell surface receptors (MDCK-SIAT1) (15, 34) or a novel cell culture-based system that morphologically and functionally recapitulates differentiated normal human bronchial epithelial (NHBE) cells (24). Investigations in vivo typically compare replication efficiencies, clinical signs, and transmissibility levels between oseltamivir-resistant viruses and the corresponding wild-type virus. Initial studies found that NA inhibitor-resistant influenza viruses were severely compromised in vitro and in animal models (6, 17, 26) and thus led to the idea that resistant viruses will unlikely have an impact on epidemic and pandemic influenza. However, clinically derived H1N1 virus with the H274Y NA mutation (18) and reverse genetics-derived H3N2 virus with the E119V NA mutation (46) were subsequently found to possess biological fitness and transmissibility similar to those of drug-sensitive virus in direct-contact ferrets. Recent studies in a guinea pig model showed that recombinant human H3N2 influenza viruses carrying either a single E119V NA mutation or the double NA mutation E119V-I222V were transmitted efficiently by direct contact but not by aerosol (5).There is limited information about the fitness of NA inhibitor-resistant H5N1 influenza viruses. Although they are not efficiently transmitted from human to human, their pandemic potential remains a serious public health concern because of their virulence in humans (1, 4, 7). H5N1 viruses isolated from untreated patients are susceptible to the NA inhibitors oseltamivir and zanamivir (21), although oseltamivir-resistant variants with the H274Y NA mutation have been reported to occur in five patients after (9, 30) or before (41) treatment with oseltamivir. The World Health Organization reported the isolation of two oseltamivir-resistant H5N1 viruses from an Egyptian girl and her uncle (44) after oseltamivir treatment. The virus was moderately resistant and possessed an N294S NA mutation. Preliminary evidence suggests that the resistance mutation existed before transmission of the virus from birds to the patients and thus before initiation of treatment (41). We previously showed that wild-type A/Vietnam/1203/04 (H5N1) influenza virus and recombinants carrying either the H274Y or the N294S NA mutation reached comparable titers in MDCK and MDCK-SIAT1 cells and caused comparable mortality rates among BALB/c mice (48). In contrast, clinically derived A/Hanoi/30408/05 (H5N1) influenza virus with the H274Y NA mutation reproduced to lower titers than the oseltamivir-sensitive virus in the lungs of inoculated ferrets (30).In a ferret model, we compared the fitness levels of two pairs of H5N1 viruses in the absence of selective drug pressure. One virus of each pair was the wild type, while the other carried the H274Y NA mutation conferring oseltamivir resistance. The two viruses used, A/Vietnam/1203/04 (HA clade 1) and A/Turkey/15/06 (HA clade 2.2), differ in their pathogenicity to ferrets. Virus fitness was evaluated by two approaches. Using the traditional approach, we compared clinical disease signs, relative inactivity indexes, weight and temperature changes, and virus replication levels in the upper respiratory tract (URT). We then used a novel competitive fitness approach in which we genetically analyzed individual virus clones after coinfection of ferrets with mixtures of oseltamivir-sensitive and -resistant H5N1 viruses; thus, we determined virus-virus interactions within the host. We observed no difference between the resistant and sensitive virus of each pair in clinical signs or virus replication in the URT; however, analysis of virus-virus interactions within the host showed that the H274Y NA mutation affected the fitness of the two viruses differently. The oseltamivir-resistant A/Vietnam/1203/04-like virus outgrew its wild-type counterpart, while the oseltamivir-resistant A/Turkey/15/06-like virus showed less fitness than its wild-type counterpart.     

Receptor Specificity of Influenza A H3N2 Viruses Isolated in Mammalian Cells and Embryonated Chicken Eggs

Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of α2-6 and α2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to α2-6- and α2-3-type receptors but retained substantial binding to specific O- and N-linked α2-3 glycans, including α2-3GalNAc and fucosylated α2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.Influenza A viruses are generally isolated and propagated in embryonated chicken eggs or in cultures of cells of mammalian origin. Human influenza viruses were previously noted to acquire mutations in the hemagglutinin (HA) gene upon isolation and culture in the allantoic sac of embryonated chicken eggs (herein simply referred to as “eggs”) compared to the sequences of those isolated in mammalian cell substrates (herein referred to as “cells”) (29, 30, 44, 53, 58). These mutations resulted in amino acid substitutions that were found to mediate receptor specificity changes and improved viral replication efficiency in eggs (37). In general, cell-grown viruses are assumed to be more similar than their egg-grown counterparts to the viruses present in respiratory secretions (30, 56). Since their emergence in 1968, influenza A (H3N2) viruses have evolved and adapted to the human host while losing their ability to be efficiently isolated and replicate in eggs, particularly after 1992 (37, 42, 48). The rate of isolation of H3N2 clinical specimens after inoculation into eggs can be up to ∼30 times lower than that in mammalian cell cultures, highlighting the strong selective pressure for the emergence of sequence variants (77).Virtually all influenza vaccines for human use were licensed decades ago by national regulatory authorities, which used a product manufactured from influenza viruses isolated and propagated exclusively in eggs; therefore, cell culture isolates have been unacceptable for this purpose (41, 71). The antigen composition of influenza vaccines requires frequent updates (every 2 years, on average) to closely match their antigenic properties to the most prevalent circulating antigenic drift variant viruses (51). The limited availability of H3N2 viruses isolated in eggs has on one or more occasions delayed vaccine composition updates and may have reduced the efficacy of vaccination against new antigenically drifted viruses (3, 34, 37).Entry of influenza viruses into host cells is mediated by HA, which binds to sialic acid containing glycoconjugates on the surface of epithelial cells in the upper respiratory tract (2, 13). The nature of the linkage between sialic acid and the vicinal sugar (usually galactose) varies in different host species and tissues and may therefore determine whether an influenza virus binds to and infects avian or human cells (40, 46, 59, 62, 72-75). Human influenza viruses preferentially bind to α2-6-linked sialic acids, and avian viruses predominantly bind to α2-3-linked sialic acids (59). Previous studies with chicken embryo chorioallantoic membranes revealed differential lectin binding, suggesting that α2-3-linked but not α2-6-linked sialosides are present on the epithelial cells (28). Human H3N2 viruses isolated in cell culture were reported to bind with a high affinity to α2-6-linked sialosides, while viruses isolated in eggs often had increased specificity for α2-3-linked sialosides (19, 20, 28). The functional classification of avian and mammalian influenza virus receptors is further complicated since in vitro and tissue-binding assays have led to new working hypotheses involving glycan chain length, topology, and the composition of the inner fragments of the carbohydrate chain as additional receptor specificity determinants (9, 17, 65, 66, 82). However, the significance of these in vitro properties remains unknown, since the structures of the natural sialosides on host cells that are used for infectious virus entry are undefined.The techniques most widely used to study the interactions of the influenza virus with host cell receptors employ animal cells in various assay formats (36, 57, 59, 64, 69). To overcome the problems of cell-based techniques, new assays that rely on labeled sialyl-glycoproteins or polymeric sialoglycans have been developed (18). However, these assays are limited by having only a few glycans available in polymeric form and offer low throughput. In contrast, glycan microarrays can assess virus binding to multiple well-defined glycans simultaneously. Previous work with influenza live or β-propiolactone (BPL)-inactivated virions as well as recombinantly produced HAs revealed a good correlation with receptor specificity compared to that achieved by other methods of analysis (4, 11, 57, 58, 65-68).Here we have compared paired isolates derived in eggs or cell cultures from the single clinical specimen to better understand their receptor binding specificity and its implications for vaccine production. We examined the differences in the sequences of the HAs between egg- and cell-grown isolates and analyzed their receptor binding profiles using glycan microarrays. Sequence analysis of the HA and glycan binding results revealed two distinct groups of viruses, with many egg isolates showing unexpectedly reduced levels of binding to α2-3 and α2-6 sialosides compared to the levels for the viruses isolated in mammalian cells. Furthermore, these studies highlighted that specific glycans may be important for H3N2 virus growth in eggs.