Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Activation Mechanisms of Natural Killer Cells during Influenza Virus Infection

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.     

Sedimentation Properties of Simian Virus 40-Specific Ribonucleic Acid Present in Green Monkey Cells During Productive Infection and in Mouse Cells Undergoing Abortive Infection

The size distribution of polyribosome-associated simian virus 40 (SV40) ribonucleic acid (RNA) was examined at various times after productive infection. Eight hours after infection, virus-specific RNA was detected in the 14 to 17S region of a sucrose gradient by deoxyribonucleic acid (DNA)-RNA hybridization; RNA present in fractions sedimenting more rapidly did not react with SV40 DNA. At successively later times, SV40 RNA was detected in more rapidly sedimenting regions. By 24 hr, a portion of the SV40 RNA was detected in the 28S region, sedimenting slightly more rapidly than a MS2 RNA marker. Nuclear SV40 RNA, prepared from cells 48 hr after infection, was distributed in more rapidly sedimenting regions of the gradient, peaking at about 32 to 34S. Some nuclear virus-specific RNA could be detected in the 45 to 50S region. During the abortive infection of mouse cells, the sedimentation profile of SV40 RNA was very similar to that observed during the early phases of the lytic cycle.     

Persistent Infection of L Cells with Vesicular Stomatitis Virus: Evolution of Virus Populations

A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37°C and that a rapid selection of RNA⁻, group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA⁻ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA⁺ mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA⁺ virus population was replaced gradually by viruses which had a ts RNA⁻ phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5°C: a defective protein (G) which renders virion infectivity heat labile at 50°C. Persistent infections initiated with this virus (ts, heat labile, RNA⁺) evolved into a virus population which was ts, heat resistant, and RNA⁻. These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA⁻ phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA⁻ mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.     

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.     

Formation of Influenza Virus Proteins

Eight virus-specific proteins have been found in chicken embryo fibroblasts infected with fowl plague virus. Among them are two glycoproteins which are the constituents of the hemagglutinin on the virus particle. They are derived from a large precursor glycoprotein by cleavage of a covalent linkage. The reaction can be blocked by the protease inhibitor diisopropylfluorophosphate and the amino acid analogue fluorophenylalanine. This indicates that a peptide bond is cleaved. If infected cells are kept at 25 C, a temperature at which virus maturation is inhibited, the precursor glycoprotein is cleaved at a significantly slower rate than at 37 C. It appears, however, that a reduced synthesis of the carbohydrate-free envelope protein is responsible for the block of virus maturation at 25 C rather than the lower cleavage rate of the precursor.     

Virus Replication in Enucleate Cells: Vesicular Stomatitis Virus and Influenza Virus

The requirement of the presence of a nucleus for the replication of vesicular stomatitis virus and influenza virus has been examined by following the growth and development of these viruses in enucleate BS-C-1 cells. Vesicular stomatitis virus replicates normally in enucleate cells with the rate of production of infectious virus, the amount of virus-specific protein synthesis, and the type of proteins produced being essentially the same in nucleate and enucleate cells. Influenza virus does not replicate in enucleate cells, no virus gene products can be detected, and there is no inhibition of cellular protein synthesis.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

Herpes Simplex Virus Products in Productive and Abortive Infection: II. Electron Microscopic and Immunological Evidence for Failure of Virus Envelopment as a Cause of Abortive Infection

Herpes simplex virus strain MPdk(-) multiplies in HEp-2 cells, but not in dog kidney (DK) cells. Strain MPdk(+)sp, a multistep mutant of MPdk(-), multiplies in both HEp-2 and DK cells. Stabilized lysates of productively infected cells yield three macromolecular aggregates of viral deoxyribonucleic acid and protein banding in CsCl gradients at densities of 1.285 g/cm(3) (alpha), 1.325 g/cm(3) (beta), and 1.37 to 1.45 g/cm(3) (gamma). Similar lysates from abortively infected cells yield only the beta and gamma bands. Electron microscopic examination revealed that (i) the alpha band contained enveloped nucleocapsids, whereas the beta band contained naked nucleocapsids and particles tentatively identified as internal components of the nucleocapsids, and that (ii) the enveloped virions and reduplication of cellular membranes observed in thin sections of productively infected cells were absent from abortively infected cells. Studies of the surface antigens of infected cells in a cytolytic system described previously revealed that abortively infected cells contained approximately 10-fold less virus-induced surface antigen than did productively infected cells. From these and other data published previously, we concluded that infectious MPdk(-) virions are not made in DK cells because (i) functional viral products necessary for the envelopment of the nucleocapsid are not made, and (ii) capsid proteins and some nonstructural products specified by the virus malfunction.     

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 Virus Infection of Marine Mammals

EcoHealth - Interspecies transmission may play a key role in the evolution and ecology of influenza A viruses. The importance of marine mammals as hosts or carriers of potential zoonotic pathogens...     

Antiviral Responses by Swine Primary Bronchoepithelial Cells Are Limited Compared to Human Bronchoepithelial Cells Following Influenza Virus Infection

Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment.     

Screening of a High Growth Influenza B Virus Strain in Vero Cells

Due to the insufficient supply of embryonated chicken eggs,the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus.The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines.However,most of the influenza viruses can not grow well in Vero cells.To develop a new influenza vaccine with Vero cells as a substrate,the virus needs to adapt to th...     

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。     

Potential Role of HPA Axis and Sympathetic Nervous Responses in Depletion of B Cells Induced by H9N2 Avian Influenza Virus Infection

Except severe pulmonary disease caused by influenza virus infection, an impaired immune system is also a clinic characteristic. However, the mechanism(s) of influenza virus infection-induced depletion of B cells was unknown. Here, we compared the effect of two variant virulence H9N2 virus infections on mouse B cells. Our study found that the infection with highly pathogenic virus (V) of led to depletion of spleen B cells and bone marrow (BM) early B cells, compared to lowly pathogenic virus (Ts). Moreover, high apoptosis and cell cycle arrest in spleen and BM were detected, suggesting important factors for the reduction of B cells in both organs. Further, this effect was not caused by virus replication in spleen and BM. Compared to Ts virus infection, V virus resulted in higher glucocorticoids (GCs) and lower leptin level in plasma. Intraperitoneal GCs receptor antagonist RU486 injection was sufficient to prevent the loss of spleen B cell and BM pro- and immature B cells, but similar result was not observed in leptin-treated mice. Depletion of spleen B cells and BM pro-B cells was also reversed by chemical sympathectomy mediated by the norepinephrine (NE) analog 6-hydroxydopamine (6-OHDA), but the treatment didn''t affect the GCs level. This study demonstrated that depletion of B cells induced by H9N2 AIV was dependent on HPA axis and sympathetic response.     

Dynamics of Influenza Virus Infection and Pathology

A key question in pandemic influenza is the relative roles of innate immunity and target cell depletion in limiting primary infection and modulating pathology. Here, we model these interactions using detailed data from equine influenza virus infection, combining viral and immune (type I interferon) kinetics with estimates of cell depletion. The resulting dynamics indicate a powerful role for innate immunity in controlling the rapid peak in virus shedding. As a corollary, cells are much less depleted than suggested by a model of human influenza based only on virus-shedding data. We then explore how differences in the influence of viral proteins on interferon kinetics can account for the observed spectrum of virus shedding, immune response, and influenza pathology. In particular, induction of high levels of interferon (“cytokine storms”), coupled with evasion of its effects, could lead to severe pathology, as hypothesized for some fatal cases of influenza.Influenza A virus causes an acute respiratory disease in humans and other mammals; in humans, it is particularly important because of the rapidity with which epidemics develop, its widespread morbidity, and the seriousness of complications. Every year, an estimated 500,000 deaths worldwide, primarily of young children and the elderly, are attributed to seasonal influenza virus infections (49). Influenza pandemics may occur when an influenza virus with new surface proteins emerges, against which the majority of the population has no preexisting immunity. Both the emergence of H5N1 virus (34) and the current H1N1 virus pandemic (43) underline the importance of understanding the dynamics of infection and disease. A key question is, what regulates virus abundance in an individual host, causing the characteristic rapid decline in virus shedding following its initial peak? The main contenders in primary influenza virus infection are depletion of susceptible target cells and the impact of the host''s innate immune response (2, 20).On infection, the influenza virus elicits an immune response, including a rapid innate response that is correlated with the observed decline in the virus load after the first 2 days of infection (1). The slower adaptive response, including both humoral and cell-mediated components, takes several days to consolidate but is important for complete virus clearance and establishment of protective immunity. During infection of an immunologically naïve host, the innate immune response is particularly important as the first line of defense against infection. The innate immune response is regulated by chemokines and cytokines, chemical messengers produced by virus-infected epithelial cells and leukocytes (23), and natural interferon-producing cells, such as plasmacytoid dendritic cells (13). Among the key cytokines induced by epithelial cells infected with influenza A virus are type I interferons (IFNs) (IFN-α/β) (23), which directly contribute to the antiviral effect on infected and neighboring cells (38).Like other viruses, influenza A viruses have evolved strategies to limit the induction of innate immune responses (38). The NS1 protein plays a dominant role, and without it, the virus is unable to grow well or to cause pathology in an immunocompetent host (14). NS1 is multifunctional and counteracts both the induction of IFN expression and the function of IFN-activated antiviral effectors via multiple mechanisms (12, 17). Individual strains of influenza A virus possess these activities to various degrees (15, 21, 22, 26), and accordingly, NS1 has been implicated as a virulence factor (3, 17). A striking effect of the failure to control the innate response to virus infection is seen as a “cytokine storm,” which causes severe pathology (8).While there is an extensive literature on modeling influenza virus spread at the population level, the individual-host scale has received much less attention (2, 4, 5, 18, 19, 20, 27, 28). In a recent important paper, Baccam et al. modeled the kinetics of influenza A virus (2). The innate dynamics were included in the form of an IFN response that delayed and reduced virus production but did not prevent it; thus, the infection was resolved primarily through near-total depletion of epithelial cells. Their model was fitted to virus titers from human volunteers exposed to H1N1 influenza virus, but no data were available on the innate immune response or epithelial cell pathology. This has been a general difficulty in developing and validating more refined within-host models; there is a lack of detailed biological data from natural host systems, in particular, measures of immune kinetics and patterns of cellular depletion.The model presented here explicitly includes the ability of IFN to induce a fully antiviral state in order to explore the relative regulatory role of innate immunity and target cell depletion. Data from experimental infections of immunologically naïve horses with an equine influenza virus (36) allowed us to calibrate our model, not only to viral kinetics, but also to IFN dynamics and cell depletion in the context of infection of a naïve natural mammalian host. With our fitted model, we then investigate modulation of the immune response.