Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells

The unparalleled spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses has resulted in devastating outbreaks in domestic poultry and sporadic human infections with a high fatality rate. To better understand the mechanism(s) of H5N1 virus pathogenesis and host responses in humans, we utilized a polarized human bronchial epithelial cell model that expresses both avian alpha-2,3- and human alpha-2,6-linked sialic acid receptors on the apical surface and supports productive replication of both H5N1 and H3N2 viruses. Using this model, we compared the abilities of selected 2004 HPAI H5N1 viruses isolated from humans and a recent human H3N2 virus to trigger the type I interferon (IFN) response. H5N1 viruses elicited significantly less IFN regulatory factor 3 (IRF3) nuclear translocation, as well as delayed and reduced production of IFN-beta compared with the H3N2 virus. Furthermore, phosphorylation of Stat2 and induction of IFN-stimulated genes (ISGs), such as MX1, ISG15, IRF7, and retinoic acid-inducible gene I, were substantially delayed and reduced in cells infected with H5N1 viruses. We also observed that the highly virulent H5N1 virus replicated more efficiently and induced a weaker IFN response than the H5N1 virus that exhibited low virulence in mammals in an earlier study. Our data suggest that the H5N1 viruses tested, especially the virus with the high-pathogenicity phenotype, possess greater capability to attenuate the type I IFN response than the human H3N2 virus. The attenuation of this critical host innate immune defense may contribute to the virulence of H5N1 viruses observed in humans.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

The continued pandemic threat posed by avian influenza viruses in Hong Kong

In 1997, a highly pathogenic avian H5N1 influenza virus was transmitted directly from live commercial poultry to humans in Hong Kong. Of the 18 people infected, six died. The molecular basis for the high virulence of this virus in mice was found to involve an amino acid change in the PB2 protein. To eliminate the source of the pathogenic virus, all birds in the Hong Kong markets were slaughtered. In 1999, another avian influenza virus of H9N2 subtype was transmitted to two children in Hong Kong. In 2000-2002, H5N1 avian viruses reappeared in the poultry markets of Hong Kong, although they have not infected humans. Continued circulation of H5N1 and other avian viruses in Hong Kong raises the possibility of future human influenza outbreaks. Moreover, the acquisition of properties of human viruses by the avian viruses currently circulating in southeast China might result in a pandemic.     

The HA and NS Genes of Human H5N1 Influenza A Virus Contribute to High Virulence in Ferrets

Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.     

Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling

NS1 (nonstructural protein 1) is an important virulence factor of the influenza A virus. We observed that NS1 proteins of the 1918 pandemic virus (A/Brevig Mission/1/18) and many avian influenza A viruses contain a consensus Src homology 3 (SH3) domain-binding motif. Screening of a comprehensive human SH3 phage library revealed the N-terminal SH3 of Crk and CrkL as the preferred binding partners. Studies with recombinant proteins confirmed avid binding of NS1 proteins of the 1918 virus and a representative avian H7N3 strain to Crk/CrkL SH3 but not to other SH3 domains tested, including p85alpha and p85beta. Endogenous CrkL readily co-precipitated NS1 from cells infected with the H7N3 virus. In transfected cells association with CrkL was observed for NS1 of the 1918 and H7N3 viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence motif. SH3 binding was dispensable for suppression of interferon-induced gene expression by NS1 but was associated with enhanced phosphatidylinositol 3-kinase signaling, as evidenced by increased Akt phosphorylation. Thus, the Spanish Flu virus resembles avian influenza A viruses in its ability to recruit Crk/CrkL to modulate host cell signaling.     

Modification of nonstructural protein 1 of influenza A virus by SUMO1

Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.     

The fight against new types of influenza virus

In 1997, during an outbreak in chickens in Hong Kong the avian H5N1 influenza virus crossed the species barrier and infected 18 people, of which 6 cases were fatal. The virus also infected wild birds and continued to circulate and mutate in geese and ducks in southeastern China. Since this occurrence, new antigenic variants that are highly pathogenic for humans as well as wild, domestic, and exotic waterfowl continue to appear in Hong Kong. This virus is spreading across Asia, and is encroaching upon Europe and other continents. Wild birds are now considered as the main reservoir of H5N1 virus. Humans become infected with this H5N1 virus usually via close contact with infected birds or a highly contaminated environment. The very low transmissibility of this virus prevented further person-to-person dissemination in spite of the complete absence of immunity in the human population to H5N1 viruses. Viruses of the H5N1 subtype are characterized by an exceptionally high pathogenicity for humans. The cause of the viral virulence is not known so far; however, several virulence factors are considered. The unprecedented capability of H5N1 viruses to kill humans intensifies the concern about its pandemic potential with catastrophic consequences. The effectiveness of existing antivirals as well as vaccines for humans and birds are reviewed.     

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine

Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1black triangle126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1black triangle126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1black triangle126 MLV as a vaccine to differentiate infected from vaccinated animals.     

Species-Specific Contribution of the Four C-Terminal Amino Acids of Influenza A Virus NS1 Protein to Virulence

Large-scale sequence analyses of influenza viruses revealed that nonstructural 1 (NS1) proteins from avian influenza viruses have a conserved C-terminal ESEV amino acid motif, while NS1 proteins from typical human influenza viruses have a C-terminal RSKV motif. To test the influence of the C-terminal domains of NS1 on the virulence of an avian influenza virus, we generated a wild-type H7N1 virus with an ESEV motif and a mutant virus with an NS1 protein containing a C-terminal RSKV motif by reverse genetics. We compared the phenotypes of these viruses in vitro in human, mouse, and duck cells as well as in vivo in mice and ducks. In human cells, the human C-terminal RSKV domain increased virus replication. In contrast, the avian C-terminal ESEV motif of NS1 increased virulence in mice. We linked this increase in pathogenicity in mice to an increase in virus replication and to a more severe lung inflammation associated with a higher level of production of type I interferons. Interestingly, the human C-terminal RSKV motif of NS1 increased viral replication in ducks. H7N1 virus with a C-terminal RSKV motif replicated to higher levels in ducks and induced higher levels of Mx, a type I interferon-stimulated gene. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.Interspecies transmission of influenza viruses can lead to the introduction of new subtypes of influenza virus into the human population (31). The emergence of a new influenza virus that is able to spread efficiently between humans can cause a pandemic, as evidenced by the recent introduction of the swine-origin 2009 A/H1N1 virus to humans (10). The spread of avian influenza A viruses from birds to humans could also lead to the introduction of a new viral subtype with pandemic potential (22). Fortunately, the efficient replication of avian influenza A viruses in humans and interhuman transmission are generally limited and require further adaptations of the virus to humans. One determinant of host adaptation lies in the receptor binding specificity of hemagglutinin (HA) (52). In addition, several reports have underlined the role of amino acid 627 of the PB2 polymerase subunit in determining viral host range and virulence (15, 36, 44, 45). Large-scale sequence analyses of viruses isolated from different bird and mammalian species have been performed in order to identify previously unrecognized determinants of host adaptation and virulence (2, 32). Those studies have identified a 4-amino-acid motif in the C-terminal domain of NS1 that could represent a previously unnoticed host adaptation motif. Indeed, the vast majority of avian influenza viruses have an NS1 protein with a C-terminal ESEV domain, while typical human viruses have a conserved RSKV domain. The conservation of these species-specific motifs in the NS1 protein despite important sequence variability in the rest of the protein suggests that these four C-terminal amino acids are under strong selection pressure in their respective natural hosts (3, 5, 25).NS1 is a multifunctional protein implicated in the regulation of viral gene expression and in the inhibition of the host antiviral response (12). In order to test the role of these newly identified NS1 domains, Jackson et al. previously introduced various C-terminal motifs into NS1 of the mouse-adapted human influenza virus A/WSN/33 strain by use of reverse genetics (24). Mice inoculated with a virus containing an avian C-terminal ESEV NS1 domain had high viral loads in the lungs and decreased survival compared to mice inoculated with a virus containing a C-terminal RSKV domain. These results showed that the C-terminal ESEV motif found in avian NS1 proteins increases virulence in mice when introduced into a human strain of influenza virus. Whether this finding also applies to avian influenza viruses remains unknown. Moreover, whether the C-terminal ESEV domain of NS1 increases replication in human cells remains unknown. Finally, how the C-terminal domains of NS1 modulate virulence in nonmammalian hosts, such as birds, is also unknown.Here, we assessed the contribution of the C-terminal domains of NS1 to the pathogenicity of an avian influenza virus. By using reverse genetics, we generated H7N1 viruses containing an NS1 protein with a C-terminal avian ESEV domain or a C-terminal human RSKV domain. The replications of these viruses in human, mouse, and duck cell were compared. In addition, we assessed their pathogenicity in mice and ducks. Our results show that the C-terminal RSKV domain increases the replication of an avian influenza virus in human cells. To our surprise, we observed that the C-terminal RSKV domain increases replication in ducks. In contrast, the C-terminal ESEV domain increases virulence in mice. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.     

Inhibition of highly pathogenic avian H5N1 influenza virus replication by RNA oligonucleotides targeting NS1 gene

H5N1 avian influenza virus (AIV) has caused widespread infections in poultry and wild birds, and has the potential to emerge as a pandemic threat to human. In order to explore novel approaches to inhibiting highly pathogenic H5N1 influenza virus infection, we have developed short RNA oligonucleotides, specific for conserved regions of the non-structural protein gene (NS1) of AIV. In vitro the hemagglutination (HA) titers in RNA oligonucleotide-treated cells were at least 5-fold lower than that of the control. In vivo, the treatment with three doses of RNA oligonucleotides protected the infected chickens from H5N1 virus-induced death at a rate of up to 87.5%. Plaque assay and real-time PCR analysis showed a significant reduction of the PFU and viral RNA level in the lung tissues of the infected animals treated with the mixed RNA oligonucleotides targeting the NS1 gene. Together, our findings revealed that the RNA oligonucleotides targeting at the AIV NS1 gene could potently inhibit avian H5N1 influenza virus reproduction and present a rationale for the further development of the RNA oligonucleotides as prophylaxis and therapy for highly pathogenic H5N1 influenza virus infection in humans.     

Influenza H5N1 and H1N1 Virus Replication and Innate Immune Responses in Bronchial Epithelial Cells Are Influenced by the State of Differentiation

Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of α2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells'' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.     

禽流感病毒最新研究进展

本文针对2004年爆发的禽流感疫病,回顾了2004年至2005年期间禽流感病毒的研究进展。逆转录聚合酶链式反应技术为禽流感病毒的分型提供了一种快速、可靠、准确的方法。对H5N1禽流感病毒致病机制的研究发现,其强致病性在于它可以躲避人类抗病毒细胞因子的作用,NS1基因编码蛋白的92位谷氨酸在其中发挥了关键作用。由于禽流感疾病多引起结膜炎,并与病毒细胞受体的研究结果相结合,有科学家认为眼部特异性是禽流感病毒的一个总体特征。社会普遍关注禽流感疫苗的研制,人类和禽类流感A型病毒M2蛋白胞外区域的序列比对工作为疫苗研制提供了一条新的思路,依据神经氨酸酶抑制剂抑制病毒的出芽繁殖原理的疫苗正在研制过程中,而利用siRNA预防和治疗禽流感也是很有潜力的一种方法。禽流感病毒研究的另一个热点是病毒基因节段的重配问题。     

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

ABSTRACT: BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quiet complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2664/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in Human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-beta mediated innate immune responses in 293T cells, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.     

Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments

Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.     

The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins

The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3' end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-β) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

Tissue tropism of swine influenza viruses and reassortants in ex vivo cultures of the human respiratory tract and conjunctiva

The 2009 pandemic influenza H1N1 (H1N1pdm) virus was generated by reassortment of swine influenza viruses of different lineages. This was the first influenza pandemic to emerge in over 4 decades and the first to occur after the realization that influenza pandemics arise from influenza viruses of animals. In order to understand the biological determinants of pandemic emergence, it is relevant to compare the tropism of different lineages of swine influenza viruses and reassortants derived from them with that of 2009 pandemic H1N1 (H1N1pdm) and seasonal influenza H1N1 viruses in ex vivo cultures of the human nasopharynx, bronchus, alveoli, and conjunctiva. We hypothesized that virus which can transmit efficiently between humans replicated well in the human upper airways. As previously reported, H1N1pdm and seasonal H1N1 viruses replicated efficiently in the nasopharyngeal, bronchial, and alveolar epithelium. In contrast, representative viruses from the classical swine (CS) (H1N1) lineage could not infect human respiratory epithelium; Eurasian avian-like swine (EA) (H1N1) viruses only infected alveolar epithelium and North American triple-reassortant (TRIG) viruses only infected the bronchial epithelium albeit inefficiently. Interestingly, a naturally occurring triple-reassortant swine virus, A/SW/HK/915/04 (H1N2), with a matrix gene segment of EA swine derivation (i.e., differing from H1N1pdm only in lacking a neuraminidase [NA] gene of EA derivation) readily infected and replicated in human nasopharyngeal and bronchial epithelia but not in the lung. A recombinant sw915 with the NA from H1N1pdm retained its tropism for the bronchus and acquired additional replication competence for alveolar epithelium. In contrast to H1N1pdm, none of the swine viruses tested nor seasonal H1N1 had tropism in human conjunctiva. Recombinant viruses generated by swapping the surface proteins (hemagglutinin and NA) of H1N1pdm and seasonal H1N1 virus demonstrated that these two gene segments together are key determinants of conjunctival tropism. Overall, these findings suggest that ex vivo cultures of the human respiratory tract provide a useful biological model for assessing the human health risk of swine influenza viruses.