Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO₄ or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.     

Viremia Associated with Fatal Outcomes in Ferrets Infected with Avian H5N1 Influenza Virus

Avian H5N1 influenza viruses cause severe disease and high mortality in infected humans. However, tissue tropism and underlying pathogenesis of H5N1 virus infection in humans needs further investigation. The objective of this work was to study viremia, tissue tropism and disease pathogenesis of H5N1 virus infection in the susceptible ferret animal model. To evaluate the relationship of morbidity and mortality with virus loads, we performed studies in ferrets infected with the H5N1 strain A/VN/1203/04 to assess clinical signs after infection and virus load in lung, brain, ileum, nasal turbinate, nasal wash, and blood. We observed that H5N1 infection in ferrets is characterized by high virus load in the brain and and low levels in the ileum using real-time PCR. In addition, viral RNA was frequently detected in blood one or two days before death and associated with symptoms of diarrhea. Our observations further substantiate pathogenicity of H5N1 and further indicate that viremia may be a bio-marker for fatal outcomes in H5N1 infection.     

The Short Stalk Length of Highly Pathogenic Avian Influenza H5N1 Virus Neuraminidase Limits Transmission of Pandemic H1N1 Virus in Ferrets

H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic, and overcoming them requires adaptations in hemagglutinin (HA) and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of highly pathogenic avian influenza (HPAI) H5N1 viruses may be the short stalk length of the neuraminidase (NA) protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study, a recombinant virus with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelial cells at 32°C. These defects correlated with a reduction in the ability of virus with a short-stalk NA to penetrate mucus and deaggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long-stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long-stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long-stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.     

Comparison of Temporal and Spatial Dynamics of Seasonal H3N2, Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Virus Infections in Ferrets

Humans may be infected by different influenza A viruses-seasonal, pandemic, and zoonotic-which differ in presentation from mild upper respiratory tract disease to severe and sometimes fatal pneumonia with extra-respiratory spread. Differences in spatial and temporal dynamics of these infections are poorly understood. Therefore, we inoculated ferrets with seasonal H3N2, pandemic H1N1 (pH1N1), and highly pathogenic avian H5N1 influenza virus and performed detailed virological and pathological analyses at time points from 0.5 to 14 days post inoculation (dpi), as well as describing clinical signs and hematological parameters. H3N2 infection was restricted to the nose and peaked at 1 dpi. pH1N1 infection also peaked at 1 dpi, but occurred at similar levels throughout the respiratory tract. H5N1 infection occurred predominantly in the alveoli, where it peaked for a longer period, from 1 to 3 dpi. The associated lesions followed the same spatial distribution as virus infection, but their severity peaked between 1 and 6 days later. Neutrophil and monocyte counts in peripheral blood correlated with inflammatory cell influx in the alveoli. Of the different parameters used to measure lower respiratory tract disease, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups. There was extra-respiratory spread to more tissues-including the central nervous system-for H5N1 infection than for pH1N1 infection, and to none for H3N2 infection. This study shows that seasonal, pandemic, and zoonotic influenza viruses differ strongly in the spatial and temporal dynamics of infection in the respiratory tract and extra-respiratory tissues of ferrets.     

Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses

Since dendritic cells may play a key role in defense against influenza virus infection, we examined the effects of recombinant hemagglutinin (HA) proteins derived from mouse-adapted H1N1 (A/WSN/1933), swine-origin 2009 pandemic H1N1 (A/Texas/05/2009), and highly pathogenic avian influenza H5N1 (A/Thailand/KAN-1/2004) viruses on mouse myeloid dendritic cells (mDCs). The results reveal that tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12) p70, and major histocompatibility complex class II (MHC-II) expression was increased in mDCs after treatment with recombinant HA proteins of H1N1 and H5N1. The specificity of recombinant HA treatments for mDC activation was diminished after proteinase K digestion. HA apparently promotes mDC maturation by enhancing CD40 and CD86 expression and suppressing endocytosis. No significant differences in mDC activation were observed among recombinant proteins of H1N1 and H5N1. The stimulation of mDCs by HA proteins of H1N1 and H5N1 was completely MyD88 dependent. These findings may provide useful information for the development of more-effective influenza vaccines.Influenza viruses trigger seasonal epidemics or pandemics of contagious diseases with mild to severe consequences in human and poultry populations worldwide (28). Members of the Orthomyxoviridae family, influenza viruses consist of single-stranded, eight-segment, negative-sense genomic RNAs, helical viral ribonucleoprotein (RNP) complexes (RNA segments, NP, PB2, PB1, and PA) and four viral envelope proteins (hemagglutinin [HA], neuraminidase [NA], and M1 and M2 matrix proteins). Type A influenza viruses are further classified into various serotypes based on the antigenic characteristics of HA and NA glycoproteins (14).In 2009, a swine-origin H1N1 strain emerged from the genetic reassortants of existing human, avian, and swine influenza viruses, resulting in a global pandemic marked by symptoms more severe than those associated with seasonal influenza virus (3, 24). According to comparative pathology in macaque monkeys, H5N1 induces greater cytokinemia, tissue damage, and interference with immune regulatory mechanisms than H1N1 infection (2). The HA spike protein of influenza virus is believed to play important roles in viral receptor binding, fusion, transmission, host range restriction, virulence, and pathogenesis (13, 27-30).Dendritic cells (DCs), considered the most potent professional antigen-presenting cells, serve as links between innate and adaptive immunity (31). Upon encountering microbial pathogens, endogenous danger signals, or inflammatory mediators, DCs mature and elicit rapid and short-lived innate immune responses before migrating to secondary lymphoid organs and enhancing adaptive immunity (17). Two major subsets of DCs are recognized in mice and humans: (i) myeloid DCs (mDCs, also called conventional DCs), which participate most directly in antigen presentation and activation of naïve T cells, and (ii) plasmacytoid DCs (pDCs), which produce type I interferons in response to viral infection (16, 42) and are also capable of inducing immunotolerance under some conditions (9). mDCs and pDCs also comprise different heterologous subsets, with unique localizations, phenotypes, and functions (36). Due to their key role in immune regulation, DCs have been developed for immunotherapeutic agents or prophylactic or therapeutic vaccines for cancer, infectious diseases, and immune system-related diseases (32, 34).DCs are essential in controlling the innate and adaptive immune responses against influenza virus infection (21). Viral RNA is recognized by various pattern recognition receptors (PRRs), including RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs). TLRs play an especially important role in detecting virus invasion and activating DCs (18, 35). However, the mechanisms causing DC activation and maturation in response to influenza viruses are not clear. HA has been described as playing an important role in modulating influenza virus virulence and host immune responses (29). In this study, we examined the effects of several recombinant HA proteins (rHAs) derived from rHA of H1N1 (rH1HA) (A/WSN/1933) and (A/Texas/05/2009) and rHA of H5N1 (rH5HA) (A/Thailand/KAN-1/2004) viruses on the activation and maturation of the mDC subset derived from mouse bone marrow.     

Oseltamivir-Resistant Pandemic A/H1N1 Virus Is as Virulent as Its Wild-Type Counterpart in Mice and Ferrets

The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK α2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets. Despite slightly reduced propagation of the mutant isolate in vitro during the first 24 h, the wild-type (WT) and mutant resistant viruses induced similar maximum weight loss in mice and ferrets with an identical pyrexic response in ferrets (AUC of 233.9 and 233.2, P = 0.5156). Similarly, comparable titers were obtained for the WT and the mutant strains on days 1, 3, 6 and 9 post-infection in mouse lungs and on days 1–7 in ferret nasal washes. A more important perivascular (day 6) and pleural (days 6 and 12) inflammation was noted in the lungs of mice infected with the H274Y mutant, which correlated with increased pulmonary levels of IL-6 and KC. Such increased levels of IL-6 were also observed in lymph nodes of ferrets infected with the mutant strain. Furthermore, the H274Y mutant strain was transmitted to ferrets. In conclusion, viral fitness of the H274Y pH1N1 isolate is not substantially altered and has the potential to induce severe disease and to disseminate.     

Neurovirulence in mice of H5N1 influenza virus genotypes isolated from Hong Kong poultry in 2001

We studied the pathogenicity of five different genotypes (A to E) of highly pathogenic avian H5N1 viruses, which contained HA genes similar to those of the H5N1 virus A/goose/Guangdong/1/96 and five different combinations of "internal" genes, in a mouse model. Highly pathogenic, neurotropic variants of genotypes A, C, D, and E were isolated from the brain after a single intranasal passage in mice. Genotype B virus was isolated from lungs only. The mouse brain variants had amino acid changes in all gene products except PB1, NP, and NS1 proteins but no common sets of mutations. We conclude that the original H5N1/01 isolates of genotypes A, C, D, and E were heterogeneous and that highly pathogenic neurotropic variants can be rapidly selected in mice.     

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.     

Influenza A Virus (H1N1) Infection Induces Glycolysis to Facilitate Viral Replication

Virologica Sinica - Viruses depend on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. In this study, we observed that influenza A...     

Permissive Replication of Homologous Murine Rotavirus in the Mouse Intestine Is Primarily Regulated by VP4 and NSP1

Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

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

Oligomeric Recombinant H5 HA1 Vaccine Produced in Bacteria Protects Ferrets from Homologous and Heterologous Wild-Type H5N1 Influenza Challenge and Controls Viral Loads Better than Subunit H5N1 Vaccine by Eliciting High-Affinity Antibodies

Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.     

糖尿病小鼠对H5N1病毒的易感性及灭活疫苗诱导的免疫反应

糖尿病患者免疫功能低下,是流感病毒感染的高危人群.研制有效的流感病毒疫苗对糖尿病患者尤为重要.以注射STZ的方法建立糖尿病小鼠模型,比较糖尿病小鼠和健康小鼠对H5N1病毒易感性的差异.病毒感染3 d后糖尿病小鼠的肺部病毒滴度比健康小鼠高,显示糖尿病小鼠对H5N1病毒更易感.用一次免疫的方法接种不同剂量的H5N1灭活疫苗(单独免疫或与佐剂共同免疫),比较其在糖尿病小鼠和健康小鼠诱导抗体应答的能力.一次免疫H5N1流感病毒灭活疫苗可诱导糖尿病小鼠产生体液免疫应答,但其抗体量低于健康小鼠,增加疫苗剂量可提高抗体水平.佐剂能增强H5N1全病毒灭活疫苗在糖尿病小鼠体内诱导的抗体反应.     

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.