In vitro growth profiles of respiratory syncytial virus in the presence of influenza virus

To elucidate epidemiological interference between respiratory syncytial (RSV) and influenza viruses, the influence of influenza A (HlN1) virus on the growth of RSV was examined. Although RSV grew in MDCK cells, coinfection with influenza A virus led to a reduction of progeny RSV. The degree of growth interference depended on the time of infection with influenza A virus post infection (p.i.) with RSV. In fact, infection with influenza A virus 12 hrs p.i. with RSV did not influence growth of the latter virus. On the contrary, growth suppression of influenza A virus by RSV was observed when the coinfection began at the later stages of RSV infection. Suppression of the growth of RSV by influenza A infection was further demonstrated at the level of viral protein synthesis. An indirect immunofluorescence (IF) test revealed that a large proportion of infected cells synthesized both RSV and influenza A virus antigens. Scanning electron microscopic (SEM) examination demonstrated that influenza A and RSV virions possessing surface antigens specific for each virus were selectively released from dually infected cells. In the present study, we proved for the first time that the growth of RSV is blocked by competitive infection with influenza A virus in a susceptible cell population, competitive protein synthesis and selective budding of RSV and influenza viruses from the same infected cells.     

NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells

Summary Purified viral preparations of influenza A virus were examined for the presence of NS₂ protein hitherto considered as a viral nonstructural protein that is present only in infected cells. Analysis of purified virus by radioimmunoprecipitation with monospecific antisera to NS₂ revealed its presence in the virus particle suggesting that it is a viral structural protein. NS₂ protein was also shown to be phosphorylated in infected cells in this study. This brings the number of influenza virus phosphoproteins to three which include NP, NS₁, and NS₂. These observations raise important questions about the role of NS₂ in the replication of influenza virus.     

Neuraminic acid is involved in the binding of influenza C virus to erythrocytes

Neuraminidases of both viral and bacterial origin have been reported to be unable to destroy the cellular receptor for influenza C virus on chicken erythrocytes, in contrast to the receptors for influenza A and B virus. However, under appropriate conditions neuraminidases from both Vibrio cholerae and Clostridium perfringens were able (i) to make chicken red blood cells resistant against agglutination by influenza C virus and (ii) to reduce the hemagglutination-inhibiting activity of rat serum. Both effects were abolished in the presence of the neuraminidase inhibitor 2,3-dehydro-2-deoxyneuraminic acid (DDN). These results indicate that contrary to previous assumptions sialic acid may very well be an essential component of the receptor for influenza C virus.     

Reproductive activity of the influenza A virus in the splenocytes of experimental animals with mono- and mixed infections

Virus-induced processes in organs and tissues of Syrian hamsters in relation to the influenza A virus strain used (HON1 or H3N2), age of the animals, and in the presence of mixed infection were compared. The infection of young hamsters with A/PR8/34 and A/Bangkok/1/79 viruses was shown to induce the synthesis of viral proteins NP and M in spleen cells lasting for up to 15 days (the observation period). In mixed influenza and respiratory syncytial virus infection the possibility of influenza virus genome expression did not change. After infection of mature hamsters, synthesis of virus-specific NP and M proteins in splenocytes was observed only in the animals infected with influenza A/PR8/34 virus but not in those infected with the less pathogenic influenza A/Bangkok/1/79 virus.     

Avian and human influenza a virus strains possess different intracellular nucleoprotein oligomerization efficiency

We have previously shown (Prokudina-Kantorovich EN and Semenova NP, Virology 223, 51-56, 1996) that the nucleoprotein (NP) of influenza A virus forms in infected cells oligomers which in the presence of SDS and 2-mercaptoethanol (ME) as reducing agent are stable at room temperature (RT) and dissociate at 100 degrees C. Here we report that the efficiency of intracellular NP oligomerization depends on the host origin of influenza A virus strain. Thus, in the cells infected with avian influenza A virus strains the viral NP was almost completely oligomerized and only traces of monomeric NP were detected by polyacrylamide gel electrophoresis (PAGE) in unboiled samples. However, in the cells infected with human influenza A virus strains, besides oligomeric NP also a significant amount of non-oligomerized monomeric NP was detected in unboiled samples. In purified virions of avian and human strains the same difference in NP monomers/oligomers ratio was detected as in the infected cells. A reassortant having all internal protein genes from a human strain and the glycoprotein genes from an avian strain revealed the same intracellular pattern of NP monomers/oligomers ratio as its parental human virus. These findings suggest that the type of NP oligomerization is controlled by the NP gene. The possible connection between the accumulation of protease-sensitive monomeric NP in cells infected with a human influenza strain and the parallel accumulation of cleaved NP in these cells is discussed.     

Host defense mechanisms against influenza virus: interaction of influenza virus with murine macrophages in vitro.

The interaction of mouse macrophages with influenza virus was examined as part of a study into the defense mechanisms against influenza infection. Macrophages exposed to A/Port Chalmers/1/73 virus produced infectious foci on susceptible indicator cell monolayers. Sampling of supernatant fluids and cells from infected macrophage cultures showed release of virus adsorbed to the cell surface. Active virus replication in macrophages could not be demonstrated. Exposing macrophages to specific antibody before or after virus infection resulted in a significant decrease in the number of infectious macrophages. The results suggest that although macrophages are not the source of replicating influenza virus, they are able to spread the infection by having virus attaching to their surface. This activity is interfered with by the presence of specific antibody.     

Enhancement of anti-influenza A virus cytotoxicity following influenza A virus vaccination in older, chronically ill adults.

We studied anti-influenza cytotoxicity by bulk peripheral blood mononuclear leukocyte (PBL) cultures derived from older, chronically ill volunteers undergoing vaccination. Vaccinees received either cold-recombinant, live-attenuated influenza A/Korea/1/82 (H3N2) virus intranasally or inactivated monovalent influenza A/Taiwan/1/86 (H1N1) subvirion vaccine intramuscularly. PBL were collected pre- and postvaccination and in vitro stimulated by autologous PBL infected with influenza A virus homologous and heterosubtypic to the respective vaccine strain. Cytotoxicity was measured against influenza A virus-infected autologous and human leukocyte antigen (HLA)-mismatched PBL targets infected with influenza A virus homologous or heterosubtypic to the vaccine virus strain. Vaccinees infected with the live-attenuated virus developed significant rises in mean anti-influenza, HLA-restricted cytotoxicity that was cross-reactive against influenza A viruses homologous and heterosubtypic to the vaccine virus. The enhanced cross-reactive cytotoxicity was inducible postvaccination by in vitro stimulation with autologous PBL infected with the homologous influenza A (H3N2) virus and with influenza A (H1N1) virus. In contrast, after vaccination with inactivated monovalent subvirion vaccine, volunteers developed significant increases in mean anti-influenza, HLA-restricted cytotoxicity only against autologous PBL infected with homologous influenza A (H1N1) virus. Increased cytotoxicity occurred only after in vitro stimulation with autologous cells infected with homologous influenza A (H1N1) virus. Mean gamma interferon levels in supernatant fluids of influenza A virus-stimulated effector PBL did not increase postvaccination, despite increased levels of anti-influenza cytotoxicity displayed by the effector cells. We conclude that the live-attenuated influenza A virus infection induced a broader range of enhanced anti-influenza cytotoxicity than did the inactivated subvirion vaccine.     

Influenza A virus infection of primary differentiated airway epithelial cell cultures derived from Syrian golden hamsters

The ability of several different influenza A virus strains to infect and replicate in primary, differentiated airway epithelial cell cultures from Syrian golden hamsters was investigated. All virus strains tested replicated equivalently in the cultures and displayed a preference for infecting nonciliated cells. This tropism correlated with the expression of both alpha2,3- and alpha2,6-linked sialic acid on the nonciliated cells. In contrast, the ciliated cells did not have detectable alpha2,6-linked sialic acid and expressed only low amounts of alpha2,3-linked sialic acid. In contrast to clinical isolates, laboratory strains of influenza A virus infected a limited number of ciliated cells at late times post-infection. The presence of alpha2,3- and alpha2,6-linked sialic acid residues on the same cell type suggests that Syrian golden hamsters and differentiated airway epithelial cell cultures derived from hamsters may provide a system for studying the reassortment of influenza A virus strains which utilize different forms of sialic acid as a primary virus receptor.     

An immunofluorescence study of influenza virus filament formation

Summary A study is described in which filamentous forms of influenza virus were observed budding from host cell surfaces. Cell cultures infected with influenza virus were stained by indirect immunofluorescence using an antiserum to purified haemagglutinin. Filaments greater than 100 µm in length, with several branch points along their length were observed; the number and length of the filaments varied according to the virus strain and the time after infection. Examination of infected cells by electron microscopy confirmed the presence of branched structures with an ultrastructure typical of filamentous forms of influenza virus. The immunofluorescence technique was quicker than thin section electron microscopy and was a more sensitive procedure for the detection of filamentous forms of influenza virus than electron microscopy using negative stain. It also enabled the antigenic composition of the filaments to be observed.With 9 Figures     

Identification of the hemagglutinating antigens of the influenza virus by immunoenzyme analysis

Highly active test sera detecting the presence of virus antigen both in concentrated and purified preparations and in allantoic virus cultures directly adsorbed on the solid phase have been proposed for successful identification and detection of influenza A and B virus variants. After direct sorption of purified and concentrated virus preparations, the test sera to influenza A (H1N1, H2N2, H3N2) virus detect the virus antigen in a concentration of 8 ng/ml, test sera to influenza B virus in a concentration of 40 ng/ml. After sorption on the solid phase of allantoic virus cultures the test sera detected influenza A virus antigen in a dose of 0.25-1 agglutinating units (AU), and antigen of influenza B virus in a dose of 1-2 AU.     

Cytotoxic T-memory cells in virus infection and the specificity of helper T cells.

Infective influenza virus primes mice and increases at least ten-fold the level of splenic cytotoxic T-memory and precursor cells in comparison with normal mice. Intranasal virus infection or intraperitoneal injection of infective virus results in frequencies of 1-2 x 10(-4) cytotoxic T-cell precursors in spleen as determined by limiting dilution assays. With both types of immunization, T-helper cells amplifying the generation of T-killer cells are limiting, and optimal clone frequencies depend on addition of excess T-helper cells. We find that at least part of the T-helper cells amplifying the generation of cytotoxic T cells are cross reactive for the type A influenza viruses and therefore have a similar virus specificity to type A influenza-specific cytotoxic T cells (tc). Help for T-killer cells can be replaced by supernatants derived from Concanavalin A-stimulated rat spleen cells, but presence of antigen is still required to stimulate the Tc precursor or memory cells before they respond to antigen non-specific T cell-growth factor(s) present in the stimulated rat spleen cell medium.     

Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-O-acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus

The receptors for the hemagglutinating encephalomyelitis virus (HEV, a porcine coronavirus) on chicken erythrocytes were analyzed and compared to the receptors for bovine coronavirus (BCV) and influenza C virus. Evidence was obtained that HEV requires the presence of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) on the cell surface for agglutination of erythrocytes as has been previously shown for BCV and influenza C virus: (i) Incubation of red blood cells with sialate 9-O-acetylesterase, the receptor-destroying enzyme of influenza C virus, rendered the erythrocytes resistant against agglutination by each of the three viruses; (ii) Human erythrocytes which are resistant to agglutination by HEV acquire receptors for HEV after resialylation with Neu5,9Ac2. Sialylation of red blood cells with limiting amounts of sialic acid indicated that strain JHB/1/66 of influenza C virus requires less Neu5,9Ac2 for agglutination of erythrocytes than the two coronaviruses, both of which were found to be similar in their reactivity with Neu5,9Ac2-containing receptors.     

High doses of recombinant mannan‐binding lectin inhibit the binding of influenza A(H1N1)pdm09 virus with cells expressing DC‐SIGN

The pandemic influenza A (H1N1)pdm09 virus continues to be a threat to human health. Low doses of mannan‐binding lectin (MBL) (<1 μg/mL) were shown not to protect against influenza A(H1N1)pdm09 infection. However, the effect of high doses of MBL has not been investigated. Dendritic cell‐specific intercellular adhesion molecule‐3 grabbing non‐integrin (DC‐SIGN) has been proposed as an alternative receptor for influenza A(H1N1)pdm09 virus. In this study, we examined the expression of DC‐SIGN on DCs as well as on acute monocytic leukemia cell line, THP‐1. High doses of recombinant or human MBL inhibited binding of influenza A(H1N1)pdm09 to both these cell types in the presence of complement derived from bovine serum. Further, anti‐DC‐SIGN monoclonal antibody inhibited binding of influenza A(H1N1)pdm09 to both DC‐SIGN‐expressing DCs and THP‐1 cells. This study demonstrates that high doses of MBL can inhibit binding of influenza A(H1N1)pdm09 virus to DC‐SIGN‐expressing cells in the presence of complement. Our results suggest that DC‐SIGN may be an alternative receptor for influenza A(H1N1)pdm09 virus.     

Characterization of human H1N1 influenza virus variants selected in vitro with zanamivir in the presence of sialic acid-containing molecules

Understanding the molecular mechanisms of influenza virus resistance to neuraminidase inhibitors is a main concern for their clinical use. In an attempt to reproduce in vivo selective conditions where influenza virus resistance to neuraminidase inhibitors can occur the zanamivir selection of an A/H1N1 influenza virus strain was carried out in Madin-Darby canine kidney cells performed in the presence or absence of sialic acid-containing inhibitor analogues that act as virus decoy receptors. The zanamivir-selected variants passaged in the presence of sialic acid-containing molecules resembling the human-like virus receptor lost the ability to bind red blood cells. Furthermore, whereas all zanamivir-selected variants exhibited a robust reduction in susceptibility to zanamivir in plaque assays only those obtained after extensive passages acquired a powerful neuraminidase enzyme resistance to zanamivir and oseltamivir. Evidence that balanced neuraminidase and hemagglutinin activities mediated by mutations induced during selection could play a role in the decrease of virus replication susceptibility to zanamivir is reported.     

Inhibitory effects of silver nanoparticles on H1N1 influenza A virus in vitro

Silver nanoparticles have demonstrated efficient inhibitory activities against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). However, the effects of silver nanoparticles against H1N1 influenza A virus remain unexplored. In this study, the interaction of silver nanoparticles with H1N1 influenza A virus was investigated. Silver nanoparticles with mean particle diameters of 10nm were prepared for the hemagglutination inhibition test, the embryo inoculation assay, and the Mosmann-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, where these tests were used to determine the inhibitory activity of silver nanoparticles on H1N1 influenza A virus. MDCK cells were used as the infection model. Electron microscopy analysis and flow cytometry assay were used to determine whether silver nanoparticles could reduce H1N1 influenza A virus-induced apoptosis in MDCK cells. This study demonstrates that silver nanoparticles have anti-H1N1 influenza A virus activities. The inhibitory effects of silver nanoparticles on influenza A virus may be a novel clinical strategy for the prevention of influenza virus infection during the early dissemination stage of the virus.     

The acquisition of anti-influenza virus activity by macrophages

Exposure of resident peritoneal macrophages or thioglycollate-induced macrophages (TG-M?) to influenza or Sendai virus-infected spleen cell culture supernatants (MAS) resulted in macrophage activation. When normal resident macrophages were used as effector cells, both infected P815 and L929 cells were lysed in the presence of MAS. MAS-activated TG-M? also lysed influenza virus-infected L929 cells. Histocompatibility between effector cells and target cells was not required for target cell destruction. The effector cells were plastic-adherent, phagocytic and Ia-. MAS-activated macrophages were also resistant to influenza virus infection in vitro. Both infectious and non-infectious preparations of influenza or Sendai virus preparations were effective at generating MAS. The mediator(s) which renders macrophages to become cytotoxic and resistant to infection was acid-stable, heat-labile (56 degrees C, 30 min; or 100 degrees C, 5 min), and the activity was neutralized by sheep antimouse type 1 interferon (IFN).     

Effects of streptovirudin on influenza viruses type A and B: inhibition of the lipid-linked oligosaccharide synthesis of fowl plague virus

Antibiotics of the streptovirudin complex (SV) inhibited the growth of influenza A and B viruses such as influenza A/fowl plague virus (FPV), strain Weybridge (Hav1 Neq1), influenza A/England 42/72 (H3N2), influenza A/Port Chalmers 1/73 (H3N2), influenza B/Leningrad 235/74, influenza B/Tokyo 7/66, and influenza B/Jamagata in chick embryo cell (CEC) cultures, in permanent canine kidney cells (MDCK), and in suspended fragments of chick embryo chorioallantoic membranes (CAM). As revealed by spectrophotometric turbidity measurements, SV completely inhibited the FPV-induced cytopathic effect (CPE). A 99.99% reduction of infectious virus yield was obtained in one-step growth cycle experiments and in the plaque reduction test. The haemagglutination inhibition titres of influenza viruses in suspended CAM fragment cultures in the presence of SV drugs were also substantially reduced. The incorporation assays indicated that SV exhibited no effect on virus-induced RNA synthesis, but influenced virus maturation by inhibition of lipid-linked oligosaccharide synthesis. A partial protection from infection was found in influenza virus A/England infected mice.     

Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.