甲1(H1N1)和甲3(H3N2)型流感病毒在小鼠体内诱导细胞免疫反应的研究

比较了甲型流感病毒A/HK/123/77(H1N1)和A/Qld/6/72(H3N2)及其具有相应表面抗原的两个冷适应基因重分配株(Cold-Adapted Reassortant)的子代毒株CR35和CR6,在小鼠体内诱导初次细胞毒性T细胞(简称Tc)反应的能力。静脉注射相同剂量病毒后第6天,甲3型A/QLd和CR6所诱导的脾细胞Tc活性均比甲1型A/HK和CR35者高100倍。编码内部蛋白的基因相同而表面抗原的基因不同的CR6与CR35所诱导的Tc活性也不同,说明了表面抗原决定着初次Tc反应的强度。用无关的流感病毒A/SW/72(H6N5)攻击已用低剂量(10~4EID_(?))病毒致敏的小鼠表明,对A/Qld和CR6致敏者所诱导的第二次Tc活性,比A/HK和CR3S致敏者的稍高;但对用大剂量病毒致敏者,则均能产生同样好的第二次Tc活性。对Tc识别感染靶细胞上的病毒成份进行了讨论。     

Role of host cytokine responses in the pathogenesis of avian H5N1 influenza viruses in mice

Highly pathogenic avian H5N1 influenza viruses are now widespread in poultry in Asia and have recently spread to some African and European countries. Interspecies transmission of these viruses to humans poses a major threat to public health. To better understand the basis of pathogenesis of H5N1 viruses, we have investigated the role of proinflammatory cytokines in transgenic mice deficient in interleukin-6 (IL-6), macrophage inflammatory protein 1 alpha (MIP-1alpha), IL-1 receptor (IL-1R), or tumor necrosis factor receptor 1 (TNFR1) by the use of two avian influenza A viruses isolated from humans, A/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486), which exhibit high and low lethality in mice, respectively. The course of disease and the extent of virus replication and spread in IL-6- and MIP-1alpha-deficient mice were not different from those observed in wild-type mice during acute infection with 1,000 50% mouse infective doses of either H5N1 virus. However, with HK/486 virus, IL-1R-deficient mice exhibited heightened morbidity and mortality due to infection, whereas no such differences were observed with the more virulent HK/483 virus. Furthermore, TNFR1-deficient mice exhibited significantly reduced morbidity following challenge with either H5N1 virus but no difference in viral replication and spread or ultimate disease outcome compared with wild-type mice. These results suggest that TNF-alpha may contribute to morbidity during H5N1 influenza virus infection, while IL-1 may be important for effective virus clearance in nonlethal H5N1 disease.     

Establishment of influenza A virus (H6N1) in minor poultry species in southern China

An H6N1 virus, A/teal/Hong Kong/W312/97 (W312), was isolated during the "bird flu" incident in Hong Kong in 1997. Genetic analysis suggested that this virus might be the progenitor of the A/Hong Kong/156/97 (HK/97) H5N1 virus, as seven of eight gene segments of those viruses had a common source. Continuing surveillance in Hong Kong showed that a W312-like virus was prevalent in quail and pheasants in 1999; however, the further development of H6N1 viruses has not been investigated since 2001. Here we report influenza virus surveillance data collected in southern China from 2000 to 2005 that show that H6N1 viruses have become established and endemic in minor poultry species and replicate mainly in the respiratory tract. Phylogenetic analysis indicated that all H6N1 isolates had W312-like hemagglutinin and neuraminidase genes. However, reassortment of internal genes between different subtype virus lineages, including H5N1, H9N2, and other avian viruses, generated multiple novel H6N1 genotypes in different types of poultry. These novel H6N1/N2 viruses are double, triple, or even quadruple reassortants. Reassortment between a W312-like H6N1 virus and an A/quail/Hong Kong/G1/97 (HK/97)-like H9N2 virus simultaneously generated novel H6N2 subtype viruses that were persistent in poultry. Molecular analyses suggest that W312-like viruses may not be the precursors of HK/97 virus but reassortants from an HK/97-like virus and another unidentified H6 subtype virus. These results provide further evidence of the pivotal role of the live poultry market system of southern China in generating increased genetic diversity in influenza viruses in this region.     

Proinflammatory Cytokine Response and Viral Replication in Mouse Bone Marrow Derived Macrophages Infected with Influenza H1N1 and H5N1 Viruses

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.     

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.     

Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses

The segmented structure of the influenza virus genome plays a pivotal role in its adaptation to new hosts and the emergence of pandemics. Despite concerns about the pandemic threat posed by highly pathogenic avian influenza H5N1 viruses, little is known about the biological properties of H5N1 viruses that may emerge following reassortment with contemporary human influenza viruses. In this study, we used reverse genetics to generate the 63 possible virus reassortants derived from H5N1 and H3N2 viruses, containing the H5N1 surface protein genes, and analyzed their viability, replication efficiency, and mouse virulence. Specific constellations of avian-human viral genes proved deleterious for viral replication in cell culture, possibly due to disruption of molecular interaction networks. In particular, striking phenotypes were noted with heterologous polymerase subunits, as well as NP and M, or NS. However, nearly one-half of the reassortants replicated with high efficiency in vitro, revealing a high degree of compatibility between avian and human virus genes. Thirteen reassortants displayed virulent phenotypes in mice and may pose the greatest threat for mammalian hosts. Interestingly, one of the most pathogenic reassortants contained avian PB1, resembling the 1957 and 1968 pandemic viruses. Our results reveal the broad spectrum of phenotypes associated with H5N1/H3N2 reassortment and a possible role for the avian PB1 in the emergence of pandemic influenza. These observations have important implications for risk assessment of H5N1 reassortant viruses detected in surveillance programs.     

Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry and humans

Since the outbreak in humans of an H5N1 avian influenza virus in Hong Kong in 1997, poultry entering the live-bird markets of Hong Kong have been closely monitored for infection with avian influenza. In March 1999, this monitoring system detected geese that were serologically positive for H5N1 avian influenza virus, but the birds were marketed before they could be sampled for virus. However, viral isolates were obtained by swabbing the cages that housed the geese. These samples, known collectively as A/Environment/Hong Kong/437/99 (A/Env/HK/437/99), contained four viral isolates, which were compared to the 1997 H5N1 Hong Kong isolates. Analysis of A/Env/HK/437/99 viruses revealed that the four isolates are nearly identical genetically and are most closely related to A/Goose/Guangdong/1/96. These isolates and the 1997 H5N1 Hong Kong viruses encode common hemagglutinin (H5) genes that have identical hemagglutinin cleavage sites. Thus, the pathogenicity of the A/Env/HK/437/99 viruses was compared in chickens and in mice to evaluate the potential for disease outbreaks in poultry and humans. The A/Env/HK/437/99 isolates were highly pathogenic in chickens but caused a longer mean death time and had altered cell tropism compared to A/Hong Kong/156/97 (A/HK/156/97). Like A/HK/156/97, the A/Env/HK/437/99 viruses replicated in mice and remained localized to the respiratory tract. However, the A/Env/HK/437/99 isolates caused only mild pathological lesions in these tissues and no clinical signs of disease or death. As a measure of the immune response to these viruses, transforming growth factor beta levels were determined in the serum of infected mice and showed elevated levels for the A/Env/HK/437/99 viruses compared to the A/HK/156/97 viruses. This study is the first to characterize the A/Env/HK/437/99 viruses in both avian and mammalian species, evaluating the H5 gene from the 1997 Hong Kong H5N1 isolates in a different genetic background. Our findings reveal that at least one of the avian influenza virus genes encoded by the 1997 H5N1 Hong Kong viruses continues to circulate in mainland China and that this gene is important for pathogenesis in chickens but is not the sole determinant of pathogenicity in mice. There is evidence that H9N2 viruses, which have internal genes in common with the 1997 H5N1 Hong Kong isolates, are still circulating in Hong Kong and China as well, providing a heterogeneous gene pool for viral reassortment. The implications of these findings for the potential for human disease are discussed.     

Distinct pathogenesis of hong kong-origin H5N1 viruses in mice compared to that of other highly pathogenic H5 avian influenza viruses

In 1997, an outbreak of virulent H5N1 avian influenza virus occurred in poultry in Hong Kong (HK) and was linked to a direct transmission to humans. The factors associated with transmission of avian influenza virus to mammals are not fully understood, and the potential risk of other highly virulent avian influenza A viruses infecting and causing disease in mammals is not known. In this study, two avian and one human HK-origin H5N1 virus along with four additional highly pathogenic H5 avian influenza viruses were analyzed for their pathogenicity in 6- to 8-week-old BALB/c mice. Both the avian and human HK H5 influenza virus isolates caused severe disease in mice, characterized by induced hypothermia, clinical signs, rapid weight loss, and 75 to 100% mortality by 6 to 8 days postinfection. Three of the non-HK-origin isolates caused no detectable clinical signs. One isolate, A/tk/England/91 (H5N1), induced measurable disease, and all but one of the animals recovered. Infections resulted in mild to severe lesions in both the upper and lower respiratory tracts. Most consistently, the viruses caused necrosis in respiratory epithelium of the nasal cavity, trachea, bronchi, and bronchioles with accompanying inflammation. The most severe and widespread lesions were observed in the lungs of HK avian influenza virus-infected mice, while no lesions or only mild lesions were evident with A/ck/Scotland/59 (H5N1) and A/ck/Queretaro/95 (H5N2). The A/ck/Italy/97 (H5N2) and the A/tk/England/91 (H5N1) viruses exhibited intermediate pathogenicity, producing mild to moderate respiratory tract lesions. In addition, infection by the different isolates could be further distinguished by the mouse immune response. The non-HK-origin isolates all induced production of increased levels of active transforming growth factor beta following infection, while the HK-origin isolates did not.     

Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern China

H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.     

H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China

The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.     

Viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza H5N1 and H1N1 viruses

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocyte-derived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.     

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.     

A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice

In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.     

Characterization of low-pathogenicity H5N1 avian influenza viruses from North America

Wild-bird surveillance in North America for avian influenza (AI) viruses with a goal of early identification of the Asian H5N1 highly pathogenic AI virus has identified at least six low-pathogenicity H5N1 AI viruses between 2004 and 2006. The hemagglutinin (HA) and neuraminidase (NA) genes from all 6 H5N1 viruses and an additional 38 North American wild-bird-origin H5 subtype and 28 N1 subtype viruses were sequenced and compared with sequences available in GenBank by phylogenetic analysis. Both HA and NA were phylogenetically distinct from those for viruses from outside of North America and from those for viruses recovered from mammals. Four of the H5N1 AI viruses were characterized as low pathogenicity by standard in vivo pathotyping tests. One of the H5N1 viruses, A/MuteSwan/MI/451072-2/06, was shown to replicate to low titers in chickens, turkeys, and ducks. However, transmission of A/MuteSwan/MI/451072-2/06 was more efficient among ducks than among chickens or turkeys based on virus shed. The 50% chicken infectious dose for A/MuteSwan/MI/451072-2/06 and three other wild-waterfowl-origin H5 viruses were also determined and were between 10(5.3) and 10(7.5) 50% egg infective doses. Finally, seven H5 viruses representing different phylogenetic clades were evaluated for their antigenic relatedness by hemagglutination inhibition assay, showing that the antigenic relatedness was largely associated with geographic origin. Overall, the data support the conclusion that North American H5 wild-bird-origin AI viruses are low-pathogenicity wild-bird-adapted viruses and are antigenically and genetically distinct from the highly pathogenic Asian H5N1 virus lineage.     

NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.     

Depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza A (H5N1) virus isolated from humans

Previously, we observed that several virulent influenza A (H5N1) viruses which caused severe or fatal disease in humans were also lethal in BALB/c mice following dissemination of the virus to solid organs, including the brain. In contrast, one particular human H5N1 virus was nonlethal in mice and showed no evidence of systemic spread. To compare H5N1 viruses of varying pathogenicity for their ability to alter the mammalian immune system, mice were infected with either influenza A/Hong Kong/483/97 (HK/483) (lethal) or A/Hong Kong/486/97 (HK/486) (nonlethal) virus and monitored for lymphocyte depletion in the blood, lungs, and lymphoid tissue. Intranasal infection with HK/483 resulted in a significant decrease in the total number of circulating leukocytes evident as early as day 2 postinfection. Differential blood counts demonstrated up to an 80% drop in lymphocytes by day 4 postinfection. In contrast, nonlethal HK/486-infected mice displayed only a transient drop of lymphocytes during the infectious period. Analysis of lung and lymphoid tissue from HK/483-infected mice demonstrated a reduction in the number of CD4(+) and CD8(+) T cells and reduced synthesis of the cytokines interleukin-1beta and gamma interferon and the chemokine macrophage inflammatory protein compared with HK/486-infected mice. In contrast, the cytokine and chemokine levels were increased in the brains of mice infected with HK/483 but not HK/486. Evidence of apoptosis in the spleen and lung of HK/483-infected mice was detected in situ, suggesting a mechanism for lymphocyte destruction. These results suggest that destructive effects on the immune system may be one factor that contributes to the pathogenesis of H5N1 viruses in mammalian hosts.     

Characterization of a human H5N1 influenza A virus isolated in 2003

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans.     

DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice

In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine’s protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.     

Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009

In 2013, three reassortant swine influenza viruses (SIVs)—two H1N2 and one H3N2—were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human‐like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human‐like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human‐lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human‐lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk.     

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells

Background

Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.

Methods

We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.

Results

We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.

Conclusion

The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.