Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus

To understand more fully the molecular events associated with highly virulent or attenuated influenza virus infections, we have studied the effects of expression of the 1918 hemagglutinin (HA) and neuraminidase (NA) genes during viral infection in mice under biosafety level 3 (agricultural) conditions. Using histopathology and cDNA microarrays, we examined the consequences of expression of the HA and NA genes of the 1918 pandemic virus in a recombinant influenza A/WSN/33 virus compared to parental A/WSN/33 virus and to an attenuated virus expressing the HA and NA genes from A/New Caledonia/20/99. The 1918 HA/NA:WSN and WSN recombinant viruses were highly lethal for mice and displayed severe lung pathology in comparison to the nonlethal New Caledonia HA/NA:WSN recombinant virus. Expression microarray analysis performed on lung tissues isolated from the infected animals showed activation of many genes involved in the inflammatory response, including cytokine, apoptosis, and lymphocyte genes that were common to all three infection groups. However, consistent with the histopathology studies, the WSN and 1918 HA/NA:WSN recombinant viruses showed increased up-regulation of genes associated with activated T cells and macrophages, as well as genes involved in apoptosis, tissue injury, and oxidative damage that were not observed in the New Caledonia HA/NA:WSN recombinant virus-infected mice. These studies document clear differences in gene expression profiles that were correlated with pulmonary disease pathology induced by virulent and attenuated influenza virus infections.     

Hemagglutinin and neuraminidase matching patterns of two influenza A virus strains related to the 1918 and 2009 global pandemics

The current pandemic influenza A (H1N1) virus has revealed a complicated reassortment of various influenza A viruses. The biological study of these viruses, especially of the viral envelope proteins hemagglutinin (HA) and neuraminidase (NA), is urgently needed for the control and prevention of H1N1 viruses. We have generated H1N1-2009 and H1N1-1918 pseudotyped particles (pp) with high infectivity. Combinations of HA1918 + NA2009 and HA2009 + NA1918 also formed infectious H1N1pps, among which the HA2009 + NA1918 combination resulted in the most highly infectious pp. Our study demonstrated that some reassortments of H1N1 viruses may hold the potential to produce higher infectivity than do their ancestors.     

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication

The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.     

H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice

Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition, and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. Primary mouse and human macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro. These results together indicate that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection.     

Seasonal trivalent inactivated influenza vaccine protects against 1918 Spanish influenza virus infection in ferrets

The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.     

Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus.

The Spanish influenza pandemic of 1918-1919 caused acute illness in 25-30% of the world's population and resulted in the death of 40 million people. The complete genomic sequence of the 1918 influenza virus will be deduced using fixed and frozen tissues of 1918 influenza victims. Sequence and phylogenetic analyses of the complete 1918 haemagglutinin (HA) and neuraminidase (NA) genes show them to be the most avian-like of mammalian sequences and support the hypothesis that the pandemic virus contained surface protein-encoding genes derived from an avian influenza strain and that the 1918 virus is very similar to the common ancestor of human and classical swine H1N1 influenza strains. Neither the 1918 HA genes nor the NA genes possessed mutations that are known to increase tissue tropicity, which accounts for the virulence of other influenza strains such as A/WSN/33 or fowl plague viruses. The complete sequence of the nonstructural (NS) gene segment of the 1918 virus was deduced and tested for the hypothesis that the enhanced virulence in 1918 could have been due to type I interferon inhibition by the NS1 protein. The results from these experiments were inconclusive. Sequence analysis of the 1918 pandemic influenza virus is allowing us to test hypotheses as to the origin and virulence of this strain. This information should help to elucidate how pandemic influenza strains emerge and what genetic features contribute to their virulence.     

Alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs

Alveolar macrophages constitutively reside in the respiratory tracts of pigs and humans. An in vivo role of alveolar macrophages in defending against influenza viruses in mice infected with a reassorted influenza virus, 1918 HA/NA:Tx/91, was reported, but there has been no report on an in vivo role of alveolar macrophages in a natural host such as a pig using currently circulating human influenza virus. Here we show that in vivo depletion of alveolar macrophages in pigs by dichloromethylene diphosphonate (MDPCL2) treatment results in 40% mortality when pigs are infected with currently circulating human H1N1 influenza viruses, while none of the infected control pigs died. All infected pigs depleted of alveolar macrophages suffered from more severe respiratory signs than infected control pigs. Induction of tumor necrosis factor alpha in the infected pigs depleted of alveolar macrophages was significantly lower than that in the lungs of infected control pigs, and the induction of interleukin-10, an immunosuppressive cytokine, significantly increased in the lungs of infected pigs depleted of alveolar macrophages compared to infected control pigs. When we measured antibody titers and CD8(+) T lymphocytes expressing gamma interferon (IFN-gamma), lower antibody titers and a lower percentage of CD8(+) T lymphocytes expressing IFN-gamma were detectable in MDPCL2-treated infected pigs than in phosphate-buffered saline- and liposome-treated and infected pigs. Taken together, our findings suggest that alveolar macrophages are essential for controlling H1N1 influenza viruses in pigs.     

季节性流感病毒 H1N1 BALB / c 鼠肺适应株的建立及其分子机制简

目的 建立季节性流感病毒H1N1的鼠肺适应株,并对适应的分子机理进行研究.方法 以病毒滴鼻感染小鼠,通过在BALB/c小鼠肺组织中连续传代,观察小鼠存活情况及肺病理改变,来获得季节性流感病毒H1N1的鼠肺适应株.结果季节性流感H1N1 A/Brisbane/59/2007病毒野生型毒株,经过在小鼠体内进行8次传代后,毒力逐渐增强,从无致病力到致死率达到100%,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因发生了3个有义突变.结论 野生季节性低致病力H1N1流感病毒可经在小鼠中经过多次传代而获得高致病力H1N1鼠肺适应株,HA蛋白89位Thr至Ile的突变对毒力的增强起决定性作用.     

N-Linked Glycosylation of the Hemagglutinin Protein Influences Virulence and Antigenicity of the 1918 Pandemic and Seasonal H1N1 Influenza A Viruses

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.     

Implication of inflammatory macrophages, nuclear receptors, and interferon regulatory factors in increased virulence of pandemic 2009 H1N1 influenza A virus after host adaptation

While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here we compared the gene expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA-CA/04 was associated with the following: (i) an early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased NF-κB activation, as well as inhibition of the negative regulator TRIM24, (ii) early and persistent infiltration of immune cells, including inflammatory macrophages, and (iii) the absence of activation of lipid metabolism later in infection, which may be mediated by inhibition of nuclear receptors, including PPARG and HNF1A and -4A, with proinflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of various pathogenicities confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following infection with lethal H1N1 r1918 influenza virus. This study links differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.     

Elicitation of anti-1918 influenza virus immunity early in life prevents morbidity and lower levels of lung infection by 2009 pandemic H1N1 influenza virus in aged mice

The Spanish influenza virus pandemic of 1918 was responsible for 40 million to 50 million deaths and is antigenically similar to the swine lineage 2009 pandemic influenza virus. Emergence of the 2009 pandemic from swine into humans has raised the possibility that low levels of cross-protective immunity to past shared epitopes could confer protection. In this study, influenza viruslike particles (VLPs) were engineered to express the hemagglutinin (HA) and genes from the 1918 influenza virus to evaluate the duration of cross-protection to the H1N1 pandemic strain by vaccinating young mice (8 to 12 weeks) and then allowing the animals to age to 20 months. This immunity was long lasting, with homologous receptor-blocking antibodies detected throughout the lifespan of vaccinated mice. Furthermore, the 1918 VLPs fully protected aged mice from 2009 pandemic H1N1 virus challenge 16 months after vaccination. Histopathological assessment showed that aged vaccinated mice had significant protection from alveolar infection but less protection of the bronchial tissue than adult vaccinated mice. Additionally, passive transfer of immune serum from aged vaccinated mice resulted in protection from death but not morbidity. This is the first report describing the lifelong duration of cross-reactive immune responses elicited by a 1918 VLP vaccine in a murine model. Importantly, these lifelong immune responses did not result in decreased total viral replication but did prevent infection of the lower respiratory tract. These findings show that immunity acquired early in life can restrict the anatomical location of influenza viral replication, rather than preventing infection, in the aged.     

一株鹅源H5N2亚型高致病性禽流感病毒的分离鉴定及生物学特性分析

分离到一株鹅源 H5N2亚型高致病性禽流感病毒,SPF鸡静脉接种致病指数为2.99,但鸭子对该病毒不敏感．病毒感染小鼠后不致病,但能够在肺内有效复制,表明其具有感染哺乳动物的潜在风险．血凝素(hemagglutinin, HA)蛋白裂解位点上插入有多个连续的碱性氨基酸(-RRRKKR-),从分子上证实这是一株高致病性禽流感病毒．核酸序列比较分析表明,分离的流感病毒HA基因与A/chicken/Hubei/489/2004 (H5N1)同源率达到99.4%,神经氨酸酶(neuraminidase, NA)基因与A/chicken/Jilin/53/01(H9N2)同源率达到99.8%;氨基酸水平上,HA与2004年分离到的A/chicken/Hubei/489/2004(H5N1)、A/swan/Guangxi/307/2004(H5N1)、A/wildduck/Guangdong/314/　2004(H5N1)和A/chicken/Henan/210/2004(H5N1)同源率均为99.3%,NA 与A/chicken/Jilin/53/01(H9N2)同源率为99.6%．进化树分析结果表明,该流感病毒分离株可能是由H5N1和H9N2两个亚型病毒重排而来．     

MicroRNA Expression and Virulence in Pandemic Influenza Virus-Infected Mice

The worst known H1N1 influenza pandemic in history resulted in more than 20 million deaths in 1918 and 1919. Although the underlying mechanism causing the extreme virulence of the 1918 influenza virus is still obscure, our previous functional genomics analyses revealed a correlation between the lethality of the reconstructed 1918 influenza virus (r1918) in mice and a unique gene expression pattern associated with severe immune responses in the lungs. Lately, microRNAs have emerged as a class of crucial regulators for gene expression. To determine whether differential expression of cellular microRNAs plays a role in the host response to r1918 infection, we compared the lung cellular “microRNAome” of mice infected by r1918 virus with that of mice infected by a nonlethal seasonal influenza virus, A/Texas/36/91. We found that a group of microRNAs, including miR-200a and miR-223, were differentially expressed in response to influenza virus infection and that r1918 and A/Texas/36/91 infection induced distinct microRNA expression profiles. Moreover, we observed significant enrichment in the number of predicted cellular target mRNAs whose expression was inversely correlated with the expression of these microRNAs. Intriguingly, gene ontology analysis revealed that many of these mRNAs play roles in immune response and cell death pathways, which are known to be associated with the extreme virulence of r1918. This is the first demonstration that cellular gene expression patterns in influenza virus-infected mice may be attributed in part to microRNA regulation and that such regulation may be a contributing factor to the extreme virulence of the r1918.H1N1 influenza A viruses continue to pose serious threats to public health, as exemplified by the ongoing 2009 H1N1 influenza pandemic. The 1918-1919 H1N1 influenza pandemic was even deadlier in comparison, causing more than 20 million deaths worldwide. The keys to unlocking the mystery of the extreme virulence of the 1918 virus were provided with the reconstruction of the virus (reconstructed 1918 influenza virus [r1918]) by reverse genetics (37). The lethality of r1918 has since been examined in both mouse and macaque models (17, 18). Unlike the nonlethal infections of some other H1N1 influenza virus strains, such as A/Texas/36/91 (Tx/91) or A/Kawasaki/173/01 (K173), the r1918 causes severe and lethal pulmonary disease. We subsequently conducted functional genomics analyses that revealed that the extreme virulence of r1918 was correlated with atypical expression of immune response-related genes, including massive induction of cellular genes related to inflammatory response and cell death pathways (17, 18). In spite of these findings, the mechanistic basis for these atypical gene expression patterns remains unknown.Cellular gene expression is a complicated process and is subject to regulation by many cellular factors. As a group of newly identified cellular regulators, microRNAs are known to regulate the expression of a large number of targets, mainly cellular genes. Through mRNA degradation or translational repression of their targets, microRNAs regulate a wide range of crucial physiologic and pathological processes. For example, miR-34a acts as a tumor suppressor by inhibiting the expression of sirt1 (40), whereas miR-21 contributes to myocardial disease by inhibiting the expression of spry1 (36). By targeting zeb1/2, the miR-200 family members play roles in maintaining the epithelial phenotype of cancer cells (27). Furthermore, Let-7s regulates the expression of hbl-1, which drives the developmental progression of epidermal stem cells (5). Cellular microRNAs also play critical roles in virus-host interactions. The cellular microRNA miR-122 is an indispensable factor in supporting hepatitis C virus (HCV) replication (16), whereas miR-196 and miR-296 substantially attenuate viral replication through type I interferon (IFN)-associated pathways in liver cells (28). Furthermore, miR-125b and miR-223 directly target human immunodeficiency virus type 1 (HIV-1) mRNA, thereby attenuating viral gene expression in resting CD4⁺ T cells (14), and miR-198 modulates HIV-1 replication indirectly by repressing the expression of ccnt1 (34), a cellular factor necessary for HIV-1 replication. More importantly, viruses may promote their life cycles by modulating the intracellular environment through actively regulating the expression of multiple cellular microRNAs. For example, human T-cell lymphotropic virus type 1 (HTLV-1) modulates the expression of a number of cellular microRNAs in order to control T-cell differentiation (3). Similarly, human cytomegalovirus (HCMV) selectively manipulates the expression of miR-100 and miR-101 to facilitate its own replication (38). In contrast, the involvement of microRNAs during influenza A virus infection or pathogenesis is largely unknown.To determine whether cellular microRNAs play a role in the host response to influenza virus infection, we performed a systematic profiling of cellular microRNAs in lung tissues from mice infected with r1918 or a nonlethal seasonal influenza virus, Tx/91 (17). We identified a group of microRNAs whose expression patterns differentiated the host response to r1918 and Tx/91 infection. We assessed the potential functions of differentially expressed microRNAs by analyzing the predicted target genes whose expression was inversely correlated with the expression of these microRNAs. Our report provides a new perspective on the contribution of microRNAs to the pathogenesis of lethal 1918 influenza virus infection.     

Neuraminidase and Hemagglutinin Matching Patterns of a Highly Pathogenic Avian and Two Pandemic H1N1 Influenza A Viruses

Background

Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies.

Methodology/Principal Findings

The hemagglutinin (HA) and neuraminidase (NA) matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains) and a highly pathogenic avian influenza A virus (H5N1) were studied using a pseudotyped particle (pp) system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005) could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern.

Conclusions/Significance

Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.     

Immunologic response to the influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. I. Studies in human vaccinees

Analysis of an earlier study of H3N2 and H7N2 inactivated influenza vaccines in schoolchildren demonstrated a greater viral neuraminidase (NA) immunogenicity of the vaccine containing the H7 hemagglutinin (HA) antigen to which they had not been primed, despite the lesser NA antigen content of that vaccine. Thus, prior experience with the influenza viral HA appeared to have a negative influence on immune response to NA, the associated external glycoprotein, presumably on the basis of intermolecular antigenic competition. In a second study, sequential immunologic response to influenza viral NA was compared in college students who were immunized with either conventional commercial vaccine or an antigenic reassortant H7N1 vaccine, and who subsequently experienced natural infection with an H1N1 influenza virus. Although both vaccines were only marginally immunogenic in inducing NA antibody response in seronegative subjects, in vaccinees initially seropositive for HA antibody significant NA antibody titer increases occurred with H7N1 vaccine. Subsequent natural infection boosted NA antibody less effectively in the population previously primed by natural infection than in initially seronegative subjects primed by H7N1 vaccination. It is suggested that primary immunization monospecific for influenza viral NA may alter the subsequent pattern of immune response to one more favorable to the induction of NA antibody when virus is encountered.     

Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection.

BALB/c mice immunized with graded doses of chromatographically purified hemagglutinin (HA) and neuraminidase (NA) antigens derived from A/Hong Kong/1/68 (H3N2) influenza virus demonstrated equivalent responses when HA-specific and NA-specific serum antibodies were measured by enzyme-linked immunosorbent assays (ELISAs). Antibody responses measured by hemagglutination inhibition or neuraminidase inhibition titrations showed similar kinetic patterns, except for more rapid decline in hemagglutination inhibition antibody. Injection of mice with either purified HA or NA resulted in immunity manifested by reduction in pulmonary virus following challenge with virus containing homologous antigens. However, the nature of the immunity induced by the two antigens differed markedly. While HA immunization with all but the lowest doses of antigen prevented manifest infection, immunization with NA was infection-permissive at all antigen doses, although reduction in pulmonary virus was proportional to the amount of antigen administered. The immunizing but infection-permissive effect of NA immunization over a wide range of doses is in accord with results of earlier studies with mice in which single doses of NA and antigenically hybrid viruses were used. The demonstrable immunogenicity of highly purified NA as a single glycoprotein without adjuvant offers a novel infection-permissive approach with potentially low toxicity for human immunization against influenza virus.     

一株H5N1亚型禽流感病毒的分离与鉴定

2004年1月湖北宜昌某鸡场暴发疫病,从该鸡场濒死鸡肺组织中分离到了一株病毒,电镜切片观察到典型的禽流感病毒粒子;采用ELISA检测禽流感抗原为阳性;RT-PCR扩增HA、NA基因并测序,经BLAST分析,HA基因与A／Goose／Guangdong／1／96(H5N1)HA基因同源性为97％;NA基因与A／Goose／Guangdong／1／96(H5N1)NA基因同源性为96％,确定该分离株为禽流感病毒H5N1亚型(A／Chicken／Yichang／Lung-1／04(H5N1))。     

Identification of a highly conserved H1 subtype-specific epitope with diagnostic potential in the hemagglutinin protein of influenza A virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ² = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs.     

Adjuvant efficacy of mOMV against avian influenza virus infection in mice

Highly pathogenic avian influenza H5N1 viruses are found chiefly in birds and have caused severe disease and death in infected humans. Development of influenza vaccines capable of inducing heterosubtypic immunity against a broad range of influenza viruses is the best option for the preparedness, since vaccination remains the principal method in controlling influenza viral infections. Here, a mOMV-adjuvanted recombinant H5N2 (rH5N2) whole virus antigen vaccine with A/Environment/Korea/W149/06(H5N1)-derived H5 HA and A/Chicken/Korea/ma116/04(H9N2)-derived N2 NA in the backbone of A/Puerto Rico/8/34(H1N1) was prepared and generated by reverse genetics. Groups of mice were vaccinated by a prime-boost regime with the rH5N2 vaccine (1.75 μg of HA with/without 10 μg mOMV or aluminum hydroxide adjuvant for comparison). At two weeks post-immunizations, vaccinated mice were challenged with lethal doses of 10^3.5 EID₅₀/ml of H5N1 or H9N2 avian influenza viruses, and were monitored for 15 days. Both mOMV- and alum-adjuvant vaccine groups had high survival rates after H5N1 infection and low levels of body weight changes compared to control groups. Interestingly, the mOMV-adjuvanted group induced better cross-reactive antibody responses serologically and promoted cross-protectivity against H5N1 and H9N2 virus challenges. Our results suggest that mOMV could be used as a vaccine adjuvant in the development of effective vaccines used to control influenza A virus transmission.     

Deletions in the neuraminidase stalk region of H2N2 and H9N2 avian influenza virus subtypes do not affect postinfluenza secondary bacterial pneumonia

We investigated the synergism between influenza virus and Streptococcus pneumoniae, particularly the role of deletions in the stalk region of the neuraminidase (NA) of H2N2 and H9N2 avian influenza viruses. Deletions in the NA stalk (ΔNA) had no effect on NA activity or on the adherence of S. pneumoniae to virus-infected human alveolar epithelial (A549) and mouse lung adenoma (LA-4) cells, although it delayed virus elution from turkey red blood cells. Sequential S. pneumoniae infection of mice previously inoculated with isogenic recombinant H2N2 and H9N2 influenza viruses displayed severe pneumonia, elevated levels of intrapulmonary proinflammatory responses, and death. No differences between the WT and ΔNA mutant viruses were detected with respect to effects on postinfluenza pneumococcal pneumonia as measured by bacterial growth, lung inflammation, morbidity, mortality, and cytokine/chemokine concentrations. Differences were observed, however, in influenza virus-infected mice that were treated with oseltamivir prior to a challenge with S. pneumoniae. Under these circumstances, mice infected with ΔNA viruses were associated with a better prognosis following a secondary bacterial challenge. These data suggest that the H2N2 and H9N2 subtypes of avian influenza A viruses can contribute to secondary bacterial pneumonia and deletions in the NA stalk may modulate its outcome in the context of antiviral therapy.