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.     

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).     

Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract

Avian H7 influenza viruses have been responsible for poultry outbreaks worldwide and have resulted in numerous cases of human infection in recent years. The high rate of conjunctivitis associated with avian H7 subtype virus infections may represent a portal of entry for avian influenza viruses and highlights the need to better understand the apparent ocular tropism observed in humans. To study this, mice were inoculated by the ocular route with viruses of multiple subtypes and degrees of virulence. We found that in contrast to human (H3N2 and H1N1) viruses, H7N7 viruses isolated from The Netherlands in 2003 and H7N3 viruses isolated from British Columbia, Canada, in 2004, two subtypes that were highly virulent for poultry, replicated to a significant titer in the mouse eye. Remarkably, an H7N7 virus, as well as some avian H5N1 viruses, spread systemically following ocular inoculation, including to the brain, resulting in morbidity and mortality of mice. This correlated with efficient replication of highly pathogenic H7 and H5 subtypes in murine corneal epithelial sheets (ex vivo) and primary human corneal epithelial cells (in vitro). Influenza viruses were labeled to identify the virus attachment site in the mouse cornea. Although we found abundant H7 virus attachment to corneal epithelial tissue, this did not account for the differences in virus replication as multiple subtypes were able to attach to these cells. These findings demonstrate that avian influenza viruses within H7 and H5 subtypes are capable of using the eye as a portal of entry.Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have resulted in over 420 documented cases of human infection to date, have generally caused acute, often severe and fatal, respiratory illness (1, 50). While conjunctivitis following infection with H5N1 or human influenza viruses has been rare, most human infections associated with H7 subtype viruses have resulted in ocular and not respiratory disease (1, 9, 37, 38). Infrequent reports of human conjunctivitis infection following exposure to H7 influenza viruses date from 1977, predominantly resulting from laboratory or occupational exposure (21, 40, 48). However, in The Netherlands in 2003, more than 80 human infections with H7N7 influenza virus occurred among poultry farmers and cullers amid widespread outbreaks of HPAI in domestic poultry; the majority of these human infections resulted in conjunctivitis (14, 20). Additionally, conjunctivitis was documented in the two human infections resulting from an H7N3 outbreak in British Columbia, Canada, in 2004, as well as in H7N3- and H7N2-infected individuals in the United Kingdom in 2006 and 2007, respectively (13, 18, 29, 46, 51). The properties that contribute to an apparent ocular tropism of some influenza viruses are currently not well understood (30).Host cell glycoproteins bearing sialic acids (SAs) are the cellular receptors for influenza viruses and can be found on epithelial cells within both the human respiratory tract and ocular tissue (26, 31, 41). Both respiratory and ocular tissues additionally secrete sialylated mucins that function in pathogen defense and protection of the epithelial surface (5, 11, 22). Within the upper respiratory tract, α2-6-linked SAs (the preferred receptor for human influenza viruses) predominate on epithelial cells (26). While α2-3-linked SAs are also present to a lesser degree on respiratory epithelial cells, this linkage is more abundantly expressed on secreted mucins (2). In contrast, α2-3-linked SAs (the preferred receptor for avian influenza viruses) are found on corneal and conjunctival epithelial cells of the human eye (31, 41), while secreted ocular mucins are abundantly composed of α2-6 SAs (5). It has been suggested that avian influenza viruses are more suited to infect the ocular surface due to their general α2-3-linked SA binding preference, but this has not been demonstrated experimentally (30).The mouse model has been used previously to study the role of ocular exposure to respiratory viruses (6, 39). In mice, ocular inoculation with an H3N2 influenza virus resulted in virus replication in nasal turbinates and lung (39), whereas ocular infection with respiratory syncytial virus (RSV) resulted in detectable virus titers in the eye and lung (6). These studies have revealed that respiratory viruses are not limited to the ocular area following inoculation at this site. However, the ability of influenza viruses to replicate specifically within ocular tissue has not been examined.Despite repeated instances of conjunctivitis associated with H7 subtype infections in humans, the reasons for this apparent ocular tropism have not been studied extensively. Here, we present a murine model to study the ability of human and avian influenza viruses to cause disease by the ocular route. We found that highly pathogenic H7 and H5 influenza viruses were capable of causing a systemic and lethal infection in mice following ocular inoculation. These highly pathogenic viruses, unlike human H3N2 and H1N1 viruses, replicated to significant titers in the mouse corneal epithelium and primary human corneal epithelial cells (HCEpiCs). Identification of viruses well suited to infecting the ocular surface is the first step in better understanding the ability of influenza viruses of multiple subtypes to use this tissue as a portal of entry.     

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.     

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.     

In Vitro Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.Influenza A virus is a negative-strand RNA virus with a segmented genome within the family of Orthomyxoviridae. Influenza A viruses are divided into subtypes based on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Currently, 16 subtypes of HA and 9 subtypes of NA have been identified in the natural reservoir of all influenza A viruses, wild aquatic birds (24). Occasionally, viruses from this reservoir cross the species barrier into mammals, including humans. When animal influenza viruses are introduced in humans, the spread of the virus is generally limited but may on occasion result in sustained human-to-human transmission. Three influenza A virus subtypes originating from the wild bird reservoir—H1, H2, and H3—have formed stable lineages in humans, starting off with a pandemic and subsequently causing yearly influenza epidemics. In the 20th century, three such pandemics have occurred, in 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2). In 2009, the swine-origin H1N1 virus caused the first influenza pandemic of the 21st century (23).Efficient human-to-human transmission is a prerequisite for any influenza A virus to become pandemic. Currently, the determinants of efficient human-to-human transmission are not completely understood. However, it is believed that a switch of receptor specificity from α2,3-linked sialic acids (SA), used by avian influenza A viruses, to α2,6-linked SA, used by human influenza viruses, is essential (6, 17, 31). It has been shown that the difference in receptor use between avian and human influenza A viruses combined with the distribution of the avian and human virus receptors in the human respiratory tract results in a different localization of virus attachment (26, 33-35). Human viruses attach more abundantly to the upper respiratory tract and trachea, whereas avian viruses predominantly attach to the lower respiratory tract (5, 33-35). Theoretically, the increased presence of virus in the upper respiratory tract, due to the specificity of human influenza A viruses for α2,6-linked SA, could facilitate efficient transmission.Since 1997, highly pathogenic avian influenza (HPAI) H5N1 virus has been circulating in Southeast Asia and has spread westward to Europe, the Middle East, and Africa, resulting in outbreaks of HPAI H5N1 virus in poultry and wild birds and sporadic human cases of infection in 15 different countries (38). The widespread, continuous circulation of the HPAI H5N1 strain has spiked fears that it may acquire specificity for α2,6-linked SA, potentially resulting in a pandemic. Given the currently high case fatality rate of HPAI H5N1 virus infection in humans of ca. 60%, the effect of such a pandemic on the human population could be devastating. In recent years, several amino acid substitutions in HA of HPAI H5N1 viruses have been described, either in virus isolates from patients or introduced experimentally, that increased the binding of the HPAI H5N1 HA to α2,6-linked SA (1, 2, 10, 14, 16, 29, 39, 40). However, none of the described substitutions conferred a full switch of receptor specificity from α2,3-linked SA to α2,6-linked SA and the substitutions were described in virus strains of different geographical origins. Furthermore, it is unknown whether these substitutions led to increased attachment of the virus to cells of the upper respiratory tract, the primary site of replication of human influenza A viruses.Here, we have introduced all of the 21 previously described amino acid substitutions or combinations thereof that changed the receptor specificity of HPAI H5N1 virus strains and six additional combinations not previously described, into HA of influenza virus A/Indonesia/5/05 (IND05). Indonesia is the country that has the highest cumulative number of human cases of HPAI H5N1 virus infection (38). The receptor specificity of 27 mutant H5N1 viruses was determined and the attachment pattern of a subset of these viruses to tissues of the respiratory tract of ferret and human was determined and compared to the attachment pattern of human influenza A virus (H3N2). Subsequently, the role of NA in efficient replication of these mutant viruses was investigated. The data presented here show that receptor specificity of HA of the IND05 virus can be changed by introducing a single amino acid substitution in the receptor-binding domain, resulting in replication competent viruses that attach abundantly to the human upper respiratory tract.     

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.     

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.     

The receptor-binding domain of influenza virus hemagglutinin produced in Escherichia coli folds into its native, immunogenic structure

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins.The genetic drift of seasonal influenza viruses and the occasional emergence of pandemic strains represent a continuing and serious burden on human health. Pandemic influenza viruses arise at irregular intervals, can infect up to 50% or more of the population, and vary in disease severity. Most notably, the H1N1 Spanish influenza pandemic of 1918 killed an estimated 20 to 50 million people worldwide, and the 1957 H2N2 Asian flu and 1968 H3N2 Hong Kong flu pandemics killed between 0.5 and 1 million people in the United States alone (30). The ongoing danger of influenza was recently emphasized by the emergence of the novel H1N1 pandemic virus from Mexico in April of 2009. The urgent need to speed up vaccine production was highlighted by this outbreak because over 340,000 confirmed cases and 4,100 deaths had occurred worldwide during the 6 months that were necessary to produce a vaccine using current procedures (39).As the major surface antigen of influenza A viruses, the hemagglutinin (HA) envelope glycoprotein is the primary source of natural immunity and the key target in vaccination. However, changes in the antigenic sites of the HA protein due to antigenic drift result in lost or diminished immunity acquired from previous infection or vaccination (35). This necessitates the production of new vaccines against seasonal influenza viruses each year. The HA protein also plays a central role in the emergence of human pandemic influenza viruses. There are 16 known antigenic subtypes of HA proteins in influenza A viruses (H1 through H16), and a pandemic occurs when an influenza virus that has an HA protein to which most of the population lacks immunity acquires the ability to be efficiently transmitted from person to person.The HA protein has multiple roles in the virus life cycle, notably receptor binding and membrane fusion. The protein is synthesized as a single precursor protein, HA₀, that trimerizes and becomes glycosylated in the endoplasmic reticulum as it traffics to the cell surface (33). The HA protein contains multiple disulfide bonds and is cleaved into a mature form consisting of two subunits, HA₁ and HA₂ (9, 18). HA₂ and the N- and C-terminal portions of HA₁ form a membrane-proximal stalk that mediates membrane fusion during viral entry (40). A receptor-binding domain (HA-RBD) forms the distal head of the molecule and is inserted into the HA₁ subunit. During virus entry, the HA-RBD engages sialic acid-containing receptors on the surface of the host cell, and the virion is subsequently internalized by endocytosis (33). Structurally and functionally, the HA-RBD is a member of the lectin superfamily, and the specificity of the binding pocket contributes to the host range of influenza viruses. For example, α(2,6)-containing sialosides are typically preferred by the HA protein from human viruses and α(2,3) sialosides by the HA proteins from avian viruses (13, 28). Upon triggering by the low-pH environment of endosomes, the HA protein undergoes an irreversible conformational change (6, 40) during which the intact HA-RBDs dissociate from the stalk of the trimer (3, 14, 19, 21). This observation, together with the manner in which the lectin-like domain is inserted as a folded module into the full-length HA protein, led us to hypothesize that the HA-RBD is able to adopt its native structure in isolation. Proper folding of the isolated HA-RBD into its native immunogenic structure has important therapeutic implications because the domain contains all of the known HA antigenic epitopes responsible for antibody recognition (5), and producing a protein-based influenza vaccine composed of isolated HA-RBD would dramatically speed up vaccine development during the early stages of a pandemic.In a recently published report, a construct of the 2009 pandemic H1N1 HA protein that encompasses the HA-RBD, designated HA_63-286-RBD, was expressed in Escherichia coli as inclusion bodies, refolded and purified, and used as a vaccine to produce immunity in ferrets (2). In this report, we show that this construct behaves as a stable, structured protein in solution, can be readily crystallized, and indeed adopts a structure that is virtually indistinguishable from that in the H1N1 HA protein ectodomain (41).     

Evolutionary Dynamics of Multiple Sublineages of H5N1 Influenza Viruses in Nigeria from 2006 to 2008

Highly pathogenic A/H5N1 avian influenza (HPAI H5N1) viruses have seriously affected the Nigerian poultry industry since early 2006. Previous studies have identified multiple introductions of the virus into Nigeria and several reassortment events between cocirculating lineages. To determine the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. We identified a major new subclade in Nigeria that is phylogenetically distinguishable from all previously identified sublineages, as well as two novel reassortment events. A detailed analysis of viral phylogeography identified two major source populations for the HPAI H5N1 virus in Nigeria, one in a major commercial poultry area (southwest region) and one in northern Nigeria, where contact between wild birds and backyard poultry is frequent. These findings suggested that migratory birds from Eastern Europe or Russia may serve an important role in the introduction of HPAI H5N1 viruses into Nigeria, although virus spread through the movement of poultry and poultry products cannot be excluded. Our study provides new insight into the genesis and evolution of H5N1 influenza viruses in Nigeria and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Nigeria.Since its emergence in 1996 in Guangdong, China, highly pathogenic avian influenza virus of the H5N1 subtype (HPAI H5N1 virus) has disseminated widely across Asia, Europe, and Africa, infecting a range of domestic and wild avian species and sporadically spilling over into humans and other mammals (4, 35). Over time, the HPAI H5N1 virus has diversified into multiple phylogenetically distinct lineages, classified as clades 0 to 9 according to the unified nomenclature system (33). The H5N1 lineage currently circulating in central Asia, the Middle East, Europe, and Africa is referred to as clade 2.2 (33) and has also been described as “EMA” or Qinghai-like in previous publications (4, 17, 27). This clade originated in April 2005 during a large outbreak of a phylogenetically distinct H5N1 virus among wild bird populations at Qinghai Lake in western China (4, 17) and rapidly spread west through central Asia and Europe, eventually reaching Africa in 2006 (27). Clade 2.2 has further diversified, forming the genetic third-order clade 2.2.1 (32) and three genetically distinct sublineages (I, II, and III) (2, 19, 28), all of which are found in Africa.Since 2006 HPAI H5N1 viruses belonging to clade 2.2 have disseminated across multiple countries in western, eastern, and northern Africa: Egypt, Niger, Cameroon, Sudan, Burkina Faso, Djibouti, Ivory Coast, Ghana, Togo, Benin, and Nigeria (2). With a large poultry industry, estimated at 140 million birds (11), Nigeria has experienced several major outbreaks of HPAI H5N1 virus, posing a serious threat to food security and public health in Africa. The first case of HPAI H5N1 virus in Nigeria (sublineage I) occurred in January 2006 in the state of Kaduna, and the virus subsequently was detected in Ghana, Burkina Faso, Ivory Coast, and Sudan (2). In February 2006 sublineage II was reported in Nigeria, and it disseminated widely across the country during 2006 and 2007, also appearing in Togo (2). Clade 2.2.1, which has been prevalent in Egypt, Israel, and the Gaza Strip from 2006 to 2008, was also detected in Nigeria in 2006 (10).By the end of 2007, outbreaks of HPAI H5N1 virus in Nigeria appeared to have been successfully controlled by measures such as “stamping out with compensation,” restrictions on movement of poultry, and enhanced surveillance (13). However, in July 2008 new cases of HPAI H5N1 from a sublineage never previously detected in Africa (sublineage III) were registered in the Nigerian states of Kano and Katsina and in live bird markets in Gombe and Kebbi states (13, 21). Hence, Nigeria is the only African country where viruses belonging to clade 2.2.1 and to three different sublineages (I, II, and III) of clade 2.2 have all been detected. At least three different reassortment events between sublineages have been documented in Nigeria. Salzberg et al. identified the first reassortant strain (which we refer to as “R1”), in which four genome segments (hemagglutinin [HA], NP, NS, and PB1) belong to sublineage I and the other four segments (NA, MP, PA, and PB2) are derived from sublineage II (27). Subsequently, phylogenetic analysis showed that a 2007 reassortant strain (which we refer to as “R3”) contained the HA and NS segments from sublineage I and the other six segments from sublineage II (19, 22). Another reassortant virus (which we refer to as “R5”) contained only the NS gene segment from sublineage I, while the other seven segments were derived from sublineage II (22).Although the genetic diversity of the Nigerian HPAI H5N1 virus population has been well characterized, including multiple introductions of the virus into Nigeria and several reassortment events, little is known about the evolutionary and population growth dynamics of the virus within Nigeria. Particularly understudied are the spatial movements of individual sublineages among Nigeria''s vast poultry population. To explore the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. Using the exact date and location of collection for each viral isolate, we inferred from their phylogenetic relationships the directionality of viral gene flow among Nigerian states and identified critical regions that are likely to serve as key sources for the H5N1 virus in Nigeria.     

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.     

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.     

Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.In April 2009, the H1N1 influenza A virus of swine origin was detected in humans in North America (9, 12, 42). Evidence for its origin came from analyses of the viral genome, with six gene segments displaying the closest resemblance to American “triple-reassortant” swine viruses and two to “Eurasian-lineage” swine viruses (13, 42). After this first detection in humans, the virus spread rapidly around the globe, starting the first influenza pandemic of the 21st century. The 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively mild, with a spectrum of disease ranging from subclinical infections or mild upper respiratory tract illness to sporadic cases of severe pneumonia and acute respiratory distress syndrome (3, 11, 27, 29, 30, 37). Overall, the case-fatality rate during the start of the pandemic was not significantly higher than in seasonal epidemics in most countries. However, a marked difference was observed in the case-fatality rate in specific age groups, with seasonal influenza generally causing highest mortality in elderly and immunocompromised individuals, and the pdmH1N1 affecting a relatively large proportion of (previously healthy) young individuals (3, 11, 27, 29, 30, 37).Determinants of influenza A virus virulence have been mapped for a wide variety of zoonotic and pandemic influenza viruses to the polymerase genes, hemagglutinin (HA), neuraminidase (NA), and nonstructural protein 1 (NS1). Such virulence-associated substitutions generally facilitate more efficient replication in humans via improved interactions with host cell factors. Since most of these virulence-associated substitutions were absent in the earliest pdmH1N1s, it has been speculated that the virus could acquire some of these mutations, potentially resulting in the emergence of more pathogenic viruses. Such virulence markers could be acquired by gene reassortment with cocirculating influenza A viruses, or by mutation. The influenza virus polymerase genes, in particular PB2, have been shown to be important determinants of the virulence of the highly pathogenic avian influenza (HPAI) H5N1 and H7N7 viruses and the transmission of the 1918 H1N1 Spanish influenza virus (17, 26, 34, 51). One of the most commonly identified virulence markers to date is E627K in PB2. The glutamic acid (E) residue is generally found in avian influenza viruses, while human viruses have a lysine (K), and this mutation was described as a determinant of host range in vitro (48). Given that all human and many zoonotic influenza viruses of the last century contained 627K, it was surprising that the pdmH1N1 had 627E. In addition, an aspartate (D)-to-asparagine (N) substitution at position 701 (D701N) of PB2 has previously been shown to expand the host range of avian H5N1 virus to mice and humans and to increase virus transmission in guinea pigs (26, 46). Like E627K, D701N was absent in the genome of pdmH1N1. Thus, the pdmH1N1 was the first known human pandemic virus with 627E and 701D, and it has been speculated that pdmH1N1 could mutate into a more virulent form by acquiring one of these mutations or both. Recently, it was shown that neither E627K nor D701N in PB2 of pdmH1N1 increased its virulence in ferrets and mice (18). The PB1-F2 protein has previously also been associated with high pathogenicity of the 1918 H1N1 and HPAI H5N1 viruses (8). The PB1-F2 protein of the pdmH1N1 is truncated due to premature stop codons. However, restoration of the PB1-F2 reading frame did not result in viruses with increased virulence (15). The NS1 protein of pdmH1N1 is also truncated due to a stop codon and, as a result, does not contain a PDZ ligand domain that is involved in cell-signaling pathways and has been implicated in the pathogenicity of 1918 H1N1 and HPAI H5N1 viruses (5, 8, 21). Surprisingly, restoration of a full-length version of the NS1 gene did not result in increased virulence in animal models (16). Mutations affecting virulence and host range have further frequently been mapped to hemagglutinin (HA) and neuraminidase (NA) in relation to their interaction with α2,3- or α2,6-linked sialic acids (SAs), the virus receptors on host cells (17, 32, 35, 50). The HA gene of previous pandemic viruses incorporated substitutions that allow efficient attachment to α2,6-SAs—the virus receptor on human cells—compared to ancestral avian viruses that attach more efficiently to α2,3-SAs (35, 47, 50).To search for mutations of potential importance to public health, numerous laboratories performed genome sequencing of pdmH1N1s, resulting in the real-time accumulation of information on emergence of potential virulence markers. Of specific interest were reports on amino acid substitutions from aspartic acid (D) to glycine (G) at position 222 (position 225 in H3) in HA of pdmH1N1. This substitution was observed in a fatal case of pdmH1N1 infection in June 2009 in the Netherlands (M. Jonges et al., unpublished data). Between July and December 2009, viruses from 11 (18%) of 61 cases with severe disease outcome in Norway have also been reported to harbor the D222G substitution upon direct sequencing of HA in clinical specimens. Such mutant viruses were not observed in any of 205 mild cases investigated, and the frequency of detection of this mutation was significantly higher in severe cases than in mild cases (23). In Hong Kong, the D222G substitution was detected in 12.5% (6) and 4.1% (31) of patients with severe disease and in 0% of patients with mild disease, in two different studies without prior propagation in embryonated chicken eggs. In addition to Norway and Hong Kong, the mutation has been detected in Brazil, Japan, Mexico, Ukraine, and the United States (56). Thus, D222G in HA could be the first identified “virulence marker” of pdmH1N1. pdmH1N1 with D222G in HA have not become widespread in the population, although they were detected in several countries. However, D222G in HA is of special interest, since it has also been described as the single change in HA between two strains of the “Spanish” 1918 H1N1 virus that differed in receptor specificity (47). Furthermore, upon propagation in embryonated chicken eggs, pdmH1N1 can acquire the mutation rapidly, presumably because it results in virus adaptation to avian (α2,3-SAs) receptors (49). The presence of the substitution in pdmH1N1s in the human population and its potential association with more severe disease prompted us to test its effect on pdmH1N1 receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models.