宠物犬和猫流感病毒感染

近十年来犬和猫流感病毒感染报道迅速增多,不仅威胁到犬和猫的健康,也对公共卫生造成了影响。自2004年首次发生H3N8亚型流感病毒感染赛犬事件以来,犬流感一直在美国的赛犬和宠物犬中流行。在韩国和我国南方的犬群中出现了因H3N2亚型禽流感感染引起的肺炎病例。亚洲和欧洲均报道了猫H5N1亚型高致病性禽流感致死性感染病例,还通过实验研究发现H5N1亚型流感病毒可在猫与猫之间水平传播。这些现象预示着流感病毒进一步获得了感染哺乳动物的能力,其公共卫生意义需引起关注。为此,本文对犬和猫流感病毒感染的流行病学、临床症状、发病机制、诊断和防控措施进行了综述。     

Influenza C Virus Infection in Rats

Four-week-old rats (WKA/Hkm strain) were infected intranasally with the Ann Arbor/1/50 strain of influenza C virus and examined for clinical symptoms, virus replication, and serum antibody response. Although the animals showed no definite signs of illness, the virus replicated in the nose, and the hemagglutination-inhibiting (HI) and neutralizing antibodies were produced in their sera. When the inoculum sizes of 10^6.2 and 10^3.2 PFU were used, virus was recovered from nasal homogenates between days 1 and 10, and serum HI antibody became detectable by 10 days after infection. The rats infected with 10^1.2 PFU of the virus continued to shed virus until as late as day 20 without producing serum HI antibody. The amount of virus recovered from the nose was not affected significantly by either sex. age, or strain of the rat except that a slower virus growth was seen in the LE strain. It was also observed that the rats, previously inoculated with 10^3.2 PFU of the virus, showed no virus shedding when reinfected 7 weeks later but produced virus though in low titers when reinfected 50 to 55 weeks later. Virus was also recovered from rats once inoculated with 10^1.2 PFU of the virus when challenged 7 weeks later. Thus repeated infections characteristic of human influenza C can be produced in rats under the restricted conditions.     

炎性体及其在流感病毒感染过程中的作用

炎性体是胞液中感受危险信号、启动介导下游免疫防御或细胞死亡（pyroptosis）的多分子复合物,是细胞内天然免疫的重要受承信号转导的中介体.炎性体识别流感病毒后诱导先天免疫反应甚至pyroptosis样细胞死亡.流感病毒高尔基体表达的M2蛋白和P2X7、ATP、ROS在炎性体的调节过程中发挥了重要作用,微生物也可以通过激活炎性体调节呼吸道粘膜免疫.炎性体的提出为最优疫苗的设计提供了新的思路.     

Role of Interleukin-12 in Primary Influenza Virus Infection

The effect of endogenous interleukin-12 (IL-12) on the influenza virus immune response in BALB/c mice was evaluated. Following primary influenza virus infection, IL-12 mRNA and protein are detected in the lung, with live virus being required for cytokine induction. Endogenous IL-12 contributes to early NK cell-dependent gamma interferon (IFN-γ) production (days 3 and 5) but not late T-cell-dependent IFN-γ secretion (day 7). IL-12 contributes to the inhibition of early virus replication but is not required for virus clearance. IL-12 also modestly contributes to the activation of cytotoxic T lymphocytes. Thus, in this model of experimental influenza virus infection, endogenous IL-12 contributes primarily to the early development and activation of the innate immune response.     

Investigating Influenza A Virus Infection: Tools To Track Infection and Limit Tropism

Influenza A viruses display a broad cellular tropism within the respiratory tracts of mammalian hosts. Uncovering the relationship between tropism and virus immunity, pathogenesis, and transmission will be critical for the development of therapeutic interventions. Here we discuss recent developments of several recombinant strains of influenza A virus. These viruses have inserted reporters to track tropism, microRNA target sites to restrict tropism, or barcodes to assess transmission dynamics, expanding our understanding of pathogen-host interactions.     

Nanodisc-Incorporated Hemagglutinin Provides Protective Immunity against Influenza Virus Infection

Every year, influenza virus infection causes significant mortality and morbidity in human populations. Although egg-based inactivated viral vaccines are available, their effectiveness depends on the correct prediction of the circulating viral strains and is limited by the time constraint of the manufacturing process. Recombinant subunit vaccines are easier to manufacture with a relatively short lead time but are limited in their efficacy partly because the purified recombinant membrane proteins in the soluble form most likely do not retain their native membrane-bound structure. Nanodisc (ND) particles are soluble, stable, and reproducibly prepared discoid shaped nanoscale structures that contain a discrete lipid bilayer bound by two amphipathic scaffold proteins. Because ND particles permit the functional reconstitution of membrane/envelope proteins, we incorporated recombinant hemagglutinin (HA) from influenza virus strain A/New Caledonia/20/99 (H1N1) into NDs and investigated their potential to elicit an immune response to HA and confer immunity to influenza virus challenge relative to the commercial vaccines Fluzone and FluMist. HA-ND vaccination induced a robust anti-HA antibody response consisting of predominantly the immunoglobulin G1 (IgG1) subclass and a high hemagglutination inhibition titer. Intranasal immunization with HA-ND induced an anti-HA IgA response in nasal passages. HA-ND vaccination conferred protection that was comparable to that of Fluzone and FluMist against challenge with influenza virus strain A/Puerto Rico/8/1934 (H1N1).The influenza A virus-type viral genome encodes 11 proteins including hemagglutinin (HA) and neuraminidase (NA). HA is important in virus transmission and is also a major determinant of host range (16). NA prevents viral aggregation and helps in the release of new viruses from the infected cell (25). These glycoproteins are the principal antigens against which humoral immune responses of the host are directed. Vaccination has been accepted as the most effective method of preventing influenza virus. Current licensed vaccines against influenza virus include conventional inactivated virus vaccine, live-attenuated vaccine, or inactivated “split-virus” vaccines, all grown in embryonated chicken eggs. Influenza virus vaccines may contain residual egg-derived antigens, which is a risk factor for persons with hypersensitivity to eggs. In the case of live-attenuated vaccines that are delivered by the mucosal route, there are several potential safety concerns including the possibility that the vaccine strain could undergo spontaneous genetic change and in a rare case of simultaneous infection with another influenza virus could undergo antigenic shift. These factors are of special concern for children and the elderly, who are the primary populations at risk for influenza virus infection (9). Therefore, there is a continuing need for developing more efficacious and safer vaccines.Apart from licensed vaccines, a number of different vaccine formulations including soluble glycoproteins, virus-like particles, and subunit vaccines (6, 9, 14) with various efficacies have been developed. Recombinant glycoprotein vaccines offer many distinct advantages, including cost, the possibility of adapting them to rapidly changing strains within a short time, and independence from egg-based formulations. In experimental setups, recombinant HA (rHA) and recombinant NA have provided protection against lethal challenge to mice (18, 27). The safety, immunogenicity, and efficacy of trivalent rHA vaccines have been established (26), and a potential trivalent HA vaccine (FluBlok; Protein Sciences Corporation) is currently in phase III clinical trials.Some rHA-based vaccines elicit high titers of anti-HA antibodies. However, these antibodies do not necessarily possess a high capacity for virus neutralization. This apparent discrepancy likely results from the use of soluble HA protein that may not accurately mimic the native structure of the membrane-embedded glycoprotein on the viral envelope for immunization. This could result in a robust antibody response with a limited ability to react with “native epitopes.” This notion is supported by data from previously reported studies that indicated that antigens expressed in their native three-dimensional conformation can elicit a more effective antibody response than proteins in their nonnative forms (19). Therefore, we investigated whether rHA presented in a lipid-bilayer-embedded formulation would elicit a potent neutralizing antibody response.The Nanodisc (ND) system was developed as a novel method for functionally reconstituting membrane proteins into soluble nanoscale lipid bilayers (3, 4, 12, 22). NDs are robust, reproducible, and monodisperse discoidal particles 5.5 nm high and nominally 10 nm in diameter that are formed via a self-assembly process. ND particles contain two copies of an alpha-helical, amphipathic protein, termed membrane scaffold protein (MSP), which encircles a lipid bilayer in a “belt-like” fashion (Fig. ​(Fig.1a).1a). A mixture of phospholipids and MSP are placed in a nonequilibrium solubilized state, for instance, using detergent or high hydrostatic pressure, and the system is then allowed to approach equilibrium by the gentle removal of the perturbant. This initiates a process of self-assembly, wherein the phospholipids and MSP find each other and generate a discoidal phospholipid bilayer encircled by the MSP. The resulting nanostructures represent a highly stable and homogeneous population with an aqueous solubility in the millimolar range (11).Open in a separate windowFIG. 1.Construction of HA-NDs. (a) Schematic showing an ND particle that contains a phospholipid bilayer encircled by membrane scaffold proteins (left) (5) and the same ND particle with an embedded transmembrane protein (right) (17). (b) HA-ND assemblies were first purified by Ni²⁺ affinity chromatography. (Top left) Silver-stained SDS-PAGE showing flowthrough, wash, and elution of HA-ND assembly mix over a Ni-nitrilotriacetic acid column (FT1 and FT2 are flowthrough, and the eluate contains the eluted protein). Arrows show the positions of the 72-kDa HA band and the 25-kDa MSPs. (Top right) Anti-HA Western blotting of the same SDS-PAGE gel. Depending on the quality of purification, a certain fraction of full-length 72-kDa rHA (HA0) can exist as proteolytically cleaved HA1 (∼50-kDa) and HA2 (∼28-kDa) subunits. (Bottom left) Ni²⁺ column eluates were further purified by SEC. Silver-stained SDS-PAGE gel shows size-based fractionation of Ni²⁺ column eluate. The numbers at the bottom correspond to the fractions collected. The MSP amounts are largest at fractions 27 to 30, showing that empty NDs eluted at those fractions. (Bottom right) Anti-HA Western blotting of the same SDS-PAGE gel showing that HA-ND assemblies eluted mainly between fractions 18 and 26. (c) Elution profile of HA-ND following SEC separation. The elution times for protein standards used for calibration are indicated at the top. The control profile for empty NDs is superimposed. HA-ND assemblies have a shorter retention time than empty NDs. inj, injection. (d) HA-ND assemblies from different SEC fractions separate as discrete-sized molecules upon native PAGE separation. Silver staining (left) and anti-HA Western blotting (right) of native PAGE gels from size exclusion fractions show different HA polymers contained in NDs. Earlier fractions are rich in higher-polymeric forms of HA, while later fractions are richer in monomeric HA. Control HA was loaded in the last well to the right in both cases.The value of the ND self-assembly process is that one can simply and reproducibly incorporate membrane proteins into these structures. This is accomplished by including the membrane protein in the initial mixture of MSP, lipid, and detergent prior to the initiation of the self-assembly process. An incorporated membrane protein then finds itself in a native-like environment with stability and activity normally found in vivo. By using phospholipids with different chemical characteristics (charge, degree of unsaturation, and length of acyl chains), the bilayer environment can be optimized to accommodate functional requirements. Furthermore, larger scaffold proteins, which in turn create a larger-diameter particle, can be employed to incorporate multimers or membrane protein complexes. Numerous membrane proteins from the three major classes-integral, tethered, and embedded (including monomers and multimers)-in the lipid bilayer environment created by NDs have been studied (2-5, 8, 10, 13, 20, 23). Since the ND system creates a stable bilayer environment that mimics that encountered by a membrane protein in the cell membrane, membrane proteins display normal folding, native ligand binding kinetics, and intact signaling activity (1, 3, 5, 8, 10, 13, 17, 23).In this study, we successfully incorporated recombinant baculovirus-derived HA into NDs (HA-ND) and compared its efficacy to induce a relevant immune response and confer protection against influenza virus challenge with those of existing licensed vaccines by using a mouse model.     

Protective Effect of Dietary Xylitol on Influenza A Virus Infection

Xylitol has been used as a substitute for sugar to prevent cavity-causing bacteria, and most studies have focused on its benefits in dental care. Meanwhile, the constituents of red ginseng (RG) are known to be effective in ameliorating the symptoms of influenza virus infection when they are administered orally for 14 days. In this study, we investigated the effect of dietary xylitol on influenza A virus infection (H1N1). We designed regimens containing various fractions of RG (RGs: whole extract, water soluble fraction, saponin and polysaccharide) and xylitol, and combination of xylitol with the RG fractions. Mice received the various combinations orally for 5 days prior to lethal influenza A virus infection. Almost all the mice died post challenge when xylitol or RGs were administered separately. Survival was markedly enhanced when xylitol was administered along with RGs, pointing to a synergistic effect. The effect of xylitol plus RG fractions increased with increasing dose of xylitol. Moreover, dietary xylitol along with the RG water soluble fraction significantly reduced lung virus titers after infection. Therefore, we suggest that dietary xylitol is effective in ameliorating influenza-induced symptoms when it is administered with RG fractions, and this protective effect of xylitol should be considered in relation to other diseases.     

Simulation and Prediction of the Adaptive Immune Response to Influenza A Virus Infection

The cellular immune response to primary influenza virus infection is complex, involving multiple cell types and anatomical compartments, and is difficult to measure directly. Here we develop a two-compartment model that quantifies the interplay between viral replication and adaptive immunity. The fidelity of the model is demonstrated by accurately confirming the role of CD4 help for antibody persistence and the consequences of immune depletion experiments. The model predicts that drugs to limit viral infection and/or production must be administered within 2 days of infection, with a benefit of combination therapy when administered early, and cytotoxic CD8 T cells in the lung are as effective for viral clearance as neutralizing antibodies when present at the time of challenge. The model can be used to investigate explicit biological scenarios and generate experimentally testable hypotheses. For example, when the adaptive response depends on cellular immune cell priming, regulation of antigen presentation has greater influence on the kinetics of viral clearance than the efficiency of virus neutralization or cellular cytotoxicity. These findings suggest that the modulation of antigen presentation or the number of lung resident cytotoxic cells and the combination drug intervention are strategies to combat highly virulent influenza viruses. We further compared alternative model structures, for example, B-cell activation directly by the virus versus that through professional antigen-presenting cells or dendritic cell licensing of CD8 T cells.Understanding how the immune system combats influenza virus infection and how the virus can affect the immune system is crucial to predicting and designing prophylactic and therapeutic strategies against the infection (58). Antigenic shift and antigenic drift alter the degree to which preexisting immunity can control the virus. These factors also influence whether different arms of the adaptive immune system can cross-react against new strains of the virus. For example, shifts of the hemagglutinin (HA) and neuraminidase (NA) protein sequences limit the ability of antibodies to neutralize new variants of the virus and may make cross-reactive T-cell responses to conserved viral proteins more important. Other viral proteins, such as NS1, affect both the induction of type I interferon as well as the susceptibility of infected cells to interferon-mediated inhibition of viral gene expression (43). The efficiencies of viral replication and cell-to-cell viral spread are altered by mutations in the viral matrix and polymerase genes, while the survival of infected cells can be altered by the viral PB1-F2 protein. These attributes are influenced by mutations in the viral matrix (50, 51) and polymerase (30, 69) genes, while the survival of infected cells can be altered by the viral PB1-F2 protein (17). The multigenic aspect of influenza virus pathogenesis makes experimental prediction difficult and time-consuming. Computer simulation tools would be useful to independently dissect the potential contribution and relative importance of each factor or to investigate unexpected scenarios that are difficult to replicate experimentally.Mathematical models and computer simulations have been widely used to study viral dynamics and immune responses to viral infections, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses (SIV), lymphocytic choriomeningitis virus (19, 55, 60, 61), and influenza A virus (3, 7, 8, 13, 34, 35, 52). More complex compartmental models of the immune system (4, 23) and models incorporating differential delay equations (21, 48, 68) have been used to better reflect the time that cells reside in a particular compartment or the duration of transit between compartments. In this study, we sought to develop a two-compartment mathematical model to assess the individual contributions of antigen presentation and activation of naïve T and B cells by antigen-presenting cells (APC), CD4 T-cell help, CD8 T-cell-mediated cytotoxicity, B cells, and antibody to control influenza A virus (IAV) infection and to explore the influence of anatomical location. We developed a model which represented published experimental findings on primary influenza virus infection. More importantly, the model was used to explore alternative structures for interactions between virus and immune cells, for example, comparing virus kinetics when antigen delivery and immune cell priming occurred through direct interaction of virus and immune cells or through a cellular intermediate. The model predicts that, under some circumstances, changes affecting antigen presentation more strongly impacted viral kinetics than other viral or immune factors (28, 73, 75, 78). This model highlights the importance of the assumptions used to synthesize a model and gaps in our understanding of the immune response regulating primary influenza virus infection. We discuss the implications of these findings for future influenza virus research and theories of influenza virus virulence based on influenza virus-immune system interactions.     

DNA疫苗预防B型流感病毒感染

用基因枪和电穿孔法将B型流感病毒DNA疫苗免疫BALB/C小鼠 ,实验证明B型流感病毒HA ,NADNA疫苗能有效抵御致死性同源B型病毒感染。同时实验表明 ,基因枪方法较电穿孔法诱导抗体水平略低。     

Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection

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Highlights? Demonstrates how a bicistronic gene can influence biological circuits ? Characterizes potency of innate immune antagonism in vivo ? Demonstrates how host factors can contribute to the timing of a virus life cycle ? Implicates NEP accumulation in the timing of IAV infection     

Dynamics of Influenza Virus and Human Host Interactions During Infection and Replication Cycle

The replication and life cycle of the influenza virus is governed by an intricate network of intracellular regulatory events during infection, including interactions with an even more complex system of biochemical interactions of the host cell. Computational modeling and systems biology have been successfully employed to further the understanding of various biological systems, however, computational studies of the complexity of intracellular interactions during influenza infection is lacking. In this work, we present the first large-scale dynamical model of the infection and replication cycle of influenza, as well as some of its interactions with the host’s signaling machinery. Specifically, we focus on and visualize the dynamics of the internalization and endocytosis of the virus, replication and translation of its genomic components, as well as the assembly of progeny virions. Simulations and analyses of the models dynamics qualitatively reproduced numerous biological phenomena discovered in the laboratory. Finally, comparisons of the dynamics of existing and proposed drugs, our results suggest that a drug targeting PB1:PA would be more efficient than existing Amantadin/Rimantaine or Zanamivir/Oseltamivir.