Influenza Virus In Cell Culture Research Articles

Replication of a Dog-Origin H6N1 Influenza Virus in Cell Culture and Mice.

The world’s first natural avian-origin H6N1 influenza A virus infection case in dogs was confirmed in Taiwan in 2014. The H6N1 virus in chickens has been endemic in Taiwan since 1972. Whether the dog H6N1 virus has interspecies transmission potential is the key issue we aim to understand. Following one virus passage in embryonated eggs and two further passages in MDCK cells, we obtained two virus derivatives, E01EE (PB1 739E and PB2 627E) and E01GK (PB1 739G and PB2 627K), respectively. The pathogenicity of E01EE and E01GK was investigated using plaque assay, growth dynamic analysis and cell viability quantification in cells from different animal species. The impact of amino acid mutation on PB1 739 and PB2 627 on viral ribonucleoprotein (RNP) activity was also analyzed. Further mouse infection experiments were performed. The results showed that both E01EE and E01GK decreased cell relative viability of canine MDCK cells, human A549 cells and chicken DF1 cells. E01Gk caused greater cellular harm in MDCK and A549 cells and had significantly higher virus titers in all of the cells compared to E01EE. The PB2 627K but not PB1 739G was the critical mutation that influenced the viral RNP activity. Both E01EE and E01GK caused mice pneumonia and considerable virus shedding, especially E01GK. This report verifies PB2 E627K mutation in virulence and spotlights the potential for the dog H6N1 virus to extend interspecies transmission.

Influenza virus segment 5 (+)RNA - secondary structure and new targets for antiviral strategies

Influenza A virus is a threat for humans due to seasonal epidemics and occasional pandemics. This virus can generate new strains that are dangerous through nucleotide/amino acid changes or through segmental recombination of the viral RNA genome. It is important to gain wider knowledge about influenza virus RNA to create new strategies for drugs that will inhibit its spread. Here, we present the experimentally determined secondary structure of the influenza segment 5 (+)RNA. Two RNAs were studied: the full-length segment 5 (+)RNA and a shorter construct containing only the coding region. Chemical mapping data combined with thermodynamic energy minimization were used in secondary structure prediction. Sequence/structure analysis showed that the determined secondary structure of segment 5 (+)RNA is mostly conserved between influenza virus type A strains. Microarray mapping and RNase H cleavage identified accessible sites for oligonucleotides in the revealed secondary structure of segment 5 (+)RNA. Antisense oligonucleotides were designed based on the secondary structure model and tested against influenza virus in cell culture. Inhibition of influenza virus proliferation was noticed, identifying good targets for antisense strategies. Effective target sites fall within two domains, which are conserved in sequence/structure indicating their importance to the virus.

Experimental model to study co-infection of human immunodeficiency virus-type 1 (HIV-1IIIB) and influenza virus in cell culture

ABSTRACTPeople living with human immunodeficiency virus (HIV) demonstrate highly expressed immune deficiency and are vulnerable to exogenous viral infections (co-infections), including influenza. We were interested in developing an in vitro model to examine HIV-1 and influenza virus (Flu) co-infection based on HIV-1 sialoglycoprotein (Sgp) synthesis and virus replication in double-infected cells. A convenient and reproducible experimental model for studying the sialylation of HIV-1 glycoprotein 120 (Gp120) by direct radioactive precursors of sialic acid synthesis, [14C]N-acetylmannosamine) in cell culture was successfully introduced for investigation of co-infection. The co-infection provoked desialylation of viral Sgps, followed initially by decreased and later on, by increased HIV replication. Monoclonal antibodies (Mab) to the main Gp120 V3 neutralization epitope but not to broadly reactive Mab against Gp120 recognize and neutralize the newly exposed epitopes. A hypothesis was outlined that exogenous neuraminidase (NA) (Flu virus) changes the configuration of HIV-1 Gp120 through desialylation resulting in the exposure of a novel antigen. The proposed model could contribute to better understanding of pathogenesis of Flu co-infection in people living with HIV. In vivo, Flu co-infection most probably leads to desialylation of both HIV and the cell surface, thus facilitating the escape of HIV from immune control. The findings described here reflect the interaction between components of two viruses – NA of Flu virus and Sgps on HIV-1 surface without considering the cell surface. The hypothesis could be extended to other exogenous co-infections with agents containing NA in HIV-infected people.

Isolation of swine influenza virus in cell cultures and embryonated chicken eggs.

Influenza virus isolation is a procedure to obtain a live and infectious virus that can be used for antigenic characterization, pathogenesis investigation, and vaccine production. Embryonated chicken egg inoculation is traditionally considered the "gold standard" method for influenza virus isolation and propagation. However, many primary cells and continuous cell lines have also been examined or developed for influenza virus isolation and replication. Specifically, swine influenza virus (SIV) isolation and propagation have been attempted and compared in embryonated chicken eggs, some primary porcine cells, and a number of continuous cell lines. Currently Madin-Darby canine kidney (MDCK) cells remain the most commonly used cell line for isolation, propagation, and titration of SIV. Virus isolation in embryonated chicken eggs or in different cell lines offers alternative approaches when SIV isolation in MDCK cells is unsuccessful. Nasal swabs, lung tissues, and oral fluids are three major specimen types for SIV isolation. In this chapter, we describe the procedures of sample processing, SIV isolation in MDCK cells and in embryonated chicken eggs, as well as methods used for confirming the virus isolation results.

DIAGNOSTIC VIROLOGIQUE DE LA GRIPPE

The authors review data concerning the classical and modern virological techniques allowing the diagnosis of influenza virus infection in laboratory. Traditional isolation methods of influenza virus in cell culture, direct and rapid detection of viral antigen by immunoassay tests, molecular biology techniques based on the detection of specific viral nucleic acids by reverse transcription polymerase chain reaction, and classical serological methods are described and their preferential indications specified.

Growth of influenza A virus is not impeded by simultaneous removal of the cholesterol-binding and acylation sites in the M2 protein

Influenza virus assembly and budding occur in the 'budozone', a coalesced raft domain in the plasma membrane. The viral transmembrane protein M2 is implicated in virus particle scission, the ultimate step in virus budding, probably by wedge-like insertion of an amphiphilic helix into the membrane. In order to do this, M2 is hypothesized to be targeted to the edge of the budozone, mediated by acylation and cholesterol binding. It was recently shown that acylation and cholesterol binding affect the membrane association of the cytoplasmic tail of M2 and targeting of the protein to coalesced rafts. This study tested whether combined removal of the acylation site (C50) and the cholesterol recognition/interaction amino acid consensus motifs (key residues Y52 and Y57) in the amphiphilic helix of M2 influenced virus formation. Recombinant influenza viruses were generated in the influenza strain A/WSN/33 background with mutations in one or both of these features. In comparison with the wild-type, all mutant viruses showed very similar growth kinetics in various cell types. Wild-type and mutant viruses differed in their relative M2 content but not regarding the major structural proteins. The morphology of the viruses was not affected by mutating M2. Moreover, wild-type and mutant viruses showed comparable competitive fitness in infected cells. Lastly, a global comparison of M2 sequences revealed that there are natural virus strains with M2 devoid of both lipid-association motifs. Taken together, these results indicate that the acylation and cholesterol-binding motifs in M2 are not crucial for the replication of influenza virus in cell culture, indicating that other factors can target M2 to the budding site.

A Drosophila Model for Genetic Analysis of Influenza Viral/Host Interactions

Influenza viruses impose a constant threat to vertebrates susceptible to this family of viruses. We have developed a new tool to study virus-host interactions that play key roles in viral replication and to help identify novel anti-influenza drug targets. Via the UAS/Gal4 system we ectopically expressed the influenza virus M2 gene in Drosophila melanogaster and generated dose-sensitive phenotypes in the eye and wing. We have confirmed that the M2 proton channel is properly targeted to cell membranes in Drosophila tissues and functions as a proton channel by altering intracellular pH. As part of the efficacy for potential anti-influenza drug screens, we have also demonstrated that the anti-influenza drug amantadine, which targets the M2 proton channel, suppressed the UAS-M2 mutant phenotype when fed to larvae. In a candidate gene screen we identified mutations in components of the vacuolar V1V0 ATPase that modify the UAS-M2 phenotype. Importantly, in this study we demonstrate that Drosophila genetic interactions translate directly to physiological requirements of the influenza A virus for these components in mammalian cells. Overexpressing specific V1 subunits altered the replication capacity of influenza virus in cell culture and suggests that drugs targeting the enzyme complex via these subunits may be useful in anti-influenza drug therapies. Moreover, this study adds credence to the idea of using the M2 "flu fly" to identify new and previously unconsidered cellular genes as potential drug targets and to provide insight into basic mechanisms of influenza virus biology.

Antiviral Properties, Metabolism, and Pharmacokinetics of a Novel Azolo-1,2,4-Triazine-Derived Inhibitor of Influenza A and B Virus Replication

Influenza viruses of types A and B cause periodic pandemics in the human population. The antiviral drugs approved to combat influenza virus infections are currently limited. We have investigated an effective novel inhibitor of human influenza A and B viruses, triazavirine [2-methylthio-6-nitro-1,2,4-triazolo[5,1-c]-1,2,4-triazine-7(4I)-one] (TZV). TZV suppressed the replication of influenza virus in cell culture and in chicken chorioallantoic membranes, and it protected mice from death caused by type A and B influenza viruses. TZV was also effective against a rimantadine-resistant influenza virus strain and against avian influenza A virus H5N1 strains. The pharmacokinetic parameters and bioavailability of TZV were calculated after the administration of TZV to rabbits. The TZV metabolite AMTZV [2-methylthio-6-amino-1,2,4-triazolo[5,1-s]-1,2,4-triazin(e)-7(4I)-one] was discovered in IAK 293T and Huh7 cell cultures, a liver homogenate, and rabbit blood after intragastric administration of TZV. AMTZV was nontoxic and inactive as an inhibitor of influenza virus in cell culture. Most likely, this metabolite is a product of TZV elimination.

Activity of Isatine-Sulfadimidine Derivatives against 2009 Pandemic H1N1 Influenza Virus in Cell Culture

The development of antiviral drugs has provided crucial new means to mitigate or relieve the debilitating effects of many viral pathogens. New classes of inhibitors are essential to combat swine influenza viral infection. A series of isatine-sulfadimidine derivatives were screened for antiviral activity against swine influenza A/California/07/2009 (H1N1) virus in Madin-Darby canine kidney (MDCK) cell culture. Cytotoxicity of the synthesized compounds was also tested in uninfected MDCK cells. All the compounds inhibit the influenza A (H1N1) in MDCK cells. The most active compounds, SPIII-5Br and SPIII-5H, inhibited virus-induced cytopathology by 50% at 27 and 30 microM, respectively, with 50% cytotoxicity occurring at a much higher dose (975-1,000 microM). The positive control compound ribavirin inhibits the replication of the virus at 18 microM and cytotoxic concentration was found to be >1,000 microM. SPIII-5Br and SPIII-5H exhibited potency in the same range as ribavirin and are suitable candidate molecules for further investigation.

Antiviral activity of garlic extract on Influenza virus

Abstract: Influenza virus is the most important cause of annual morbidities and mortalities worldwide with numerous antigenic drifts and shifts. Inaccessibility to effective drugs and vaccines has made world health authorities to be interested in traditional medicine in order to prevent spread of the infectious agent. Garlic is one of the most famous of all plants in human history. It has been shown that garlic extract has various effects on different diseases. The aim of this study was to evaluate garlic extract antiviral activity against influenza virus in cell culture. To study the potential antiviral activity, MDCK (Madin-Darbey Canin Kidney) cells were treated with effective minimal cytotoxic concentration of the extract and 100 TCID50 (50% Tissue Culture Infectious Dose) of the virus during infection at different time periods. The viral titers were determined by hemagglutination (HA) and TCID50 assays. The antiviral effect of the extract was studied at 1, 8 and 24 hours after treatment on the culture. To measure the amount of the viral genome synthesized at different times after treatment, RNA extraction, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and free band densitometry software were performed. Although the precise mechanism has not been defined yet, it was found that garlic extract with a good selectivity index (SI) has inhibitory effect on the virus penetration and proliferation in cell culture.

Hsp90 inhibitors reduce influenza virus replication in cell culture

The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1–2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.

Biologically Active Constituents of a Polyphenol Extract from Geranium sanguineum L. with Anti-Influenza Activity

From the aerial roots of the medicinal plant Geranium sanguineum L. a polyphenol-rich extract with strong anti-influenza activity has been isolated. To investigate its active fractions, the extract was partitioned by solvents with increasing polarity. The n-BuOH fraction contained the majority of the in vitro antiviral activity; the EtOAc fraction was the most effective one in vivo. A bioassay-directed fractionation of the n-BuOH and EtOAc fractions was performed to obtain information about the nature of the chemical components of the plant extract, responsible for the antiviral effect. The individual constituents were identified by spectroscopic methods and comparison with authentic samples and by HPLC. The cell-toxic and virus-inhibitory effects of the fractions and some individual polyphenol compounds, found in Geranium sanguineum L., were studied using the replication of representative influenza viruses in cell cultures. This study showed that the presence of a variety of biologically active compounds as well as the possible synergistic interactions between them seem to be decisive for the overall antiviral effect.

In vitro characterization of A-315675, a highly potent inhibitor of A and B strain influenza virus neuraminidases and influenza virus replication.

A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (K(i)) values between 0.024 and 0.31 nM. These values were comparable to or lower than the K(i) values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections.

Phenotypic mixing with recombinant haemagglutinin of high cleavability mediates multi-cycle replication of human influenza virus in cell culture.

When CV-1 cells expressing haemagglutinin (HA) of fowl plague virus A/FPV/34/Rostock(H7) (FPV) from an SV40-based recombinant vector were superinfected with the human influenza virus A/FM/1/47(H1N1)(FM1), phenotypically mixed progeny virus was observed. It contained cleaved FPV HA and uncleaved FM1 HA, was infectious without trypsin treatment and its infectivity was neutralizable by anti-FPV serum. When superinfection of H7 HA-expressing CV-1 cells was performed at a low multiplicity of infection, multi-cycle replication occurred. Control cells preinfected with an SV40-based recombinant not expressing FPV HA did not allow multi-cycle replication. Multi-cycle replication of FM1 virus was also observed when cells were preinfected with a vector expressing a highly cleavable mutant of influenza virus A/Port Chalmers/1/73(H3) HA carrying an insert of four arginine residues at the cleavage site. This was not the case when cells expressing uncleaved wild-type H3 HA were used. The results show that by phenotypic mixing with recombinant HA of high cleavability, a human influenza virus can be obtained in infectious form from cells lacking a suitable protease to activate this virus.

Enhanced Isolation of Influenza Virus in Conventional Plate Cell Cultures by Using Low-Speed Centrifugation from Clinical Specimens

To determine whether low-speed centrifugation as a means of amplifying viral adsorption to target cells can enhance the sensitivity of conventional techniques for the isolation of influenza virus from clinical specimens, the authors conducted a simultaneous comparison between two conventional plate cell cultures--one with and one without centrifugation (700 X g, 60 minutes). Of 26 influenza virus isolates obtained from 528 clinical specimens, 17 were more efficiently isolated by the centrifugation assay compared with conventional culture methods. Centrifugation-assisted methods were found to be efficient in the recovery of 17 of 20 isolates (85%) not identified on first passage by conventional techniques. The number of isolates obtained with centrifugation was 1.5 times as high as that obtained with no centrifugation after three passages. The two assays were significantly different in the isolation of influenza virus (P less than 0.01). Since low-speed centrifugation may increase the infectivity of influenza virus in cell culture, this technique may prove useful in the efficient and rapid isolation of this virus from clinical specimens.

Rapid detection of influenza virus in cell culture by indirect immunoperoxidase staining with type-specific monoclonal antibodies.

Seventy respiratory specimens culture-positive for influenza virus were stored at -70 degrees C and retested in duplicate by virus isolation and by indirect immunoperoxidase (IPA) staining of cell monolayers 24 hr postinoculation using influenza type-specific monoclonal antibodies. The IPA stain was positive for 24 of 28 specimens from which influenza virus was reisolated and for eight specimens from which influenza virus was not reisolated.

Subtype-specific identification of influenza virus in cell cultures with FITC labelled egg yolk antibodies

We report on results obtained with a direct immunofluorescence test for subtype-specific identification of influenza virus in detached cells of MDCK cultures after inoculation of 281 clinical specimens from patients with influenza-like disease. Influenza virus antibodies were produced in eggs from immunized hens and labelled with FITC. In 157 cases CPE was found in MDCK cells. A total of 57 cases of influenza A (H3N2), 86 cases of influenza A (H1N1), and 14 cases of influenza B were identified. In 33 cases of influenza A (H1N1) infection with massive CPE guinea pig but not chicken erythrocytes were agglutinated by the cell culture supernatants. The single step immunofluorescence test described proved easy to perform and results were obtained within 1 h after CPE was observed in contrast to the conventional HIT which is very time-consuming.

Fluorometric assay for measurement of viral neuraminidase--application to the rapid detection of influenza virus in nasal wash specimens.

A sensitive assay was developed for the measurement of neuraminidase with use of the 4-methylumbelliferkyl-alph-ketoside of N-acetyl-neuraminic acid as a fluorescent substrate. Neuraminidase activity was detected in preparation containing small quantities of cultivated influenza virus as well as in some nasal wash specimens from human volunteers experimentally infected with influenza A/Alaska/78 virus. Activity due to influenza viral neuraminidase could be distinguished from activity due to bacterial or mammalian (fibroblast) neuraminidase by means of enzyme inhibition with ethylenediaminetetraacetate, a chelating agent. The same substrate could be used in a solid-phase assay to determine the antigenic type of the influenza viral neuraminidase. The fluorescent neuraminidase assay takes 2-4 hr to perform and uses equipment available in many laboratories. Thus, it offers promise for the rapid detection of influenza viruses in cell cultures and clinical specimens.

Host defense mechanisms against infection with influenza virus. I. Effect of sensitized spleen cells on infection in vitro.

Sensitized murine spleen cells decrease the spread of influenza infection in cell cultures derived from human and syngeneic murine tissues. This control of infection is immune-specific and can be induced by immunization with identical hemagglutinin or neuraminidase antigens. The antiviral effect is evident in the absence of added immune serum and can be blocked by adsorption of sensitized spleen cells with antiserum to murine IgG. The antiviral effect persists despite separation by agar of the sensitized spleen cells from the virus-infected monolayer. Spleen cells that do not adhere to glass are quantitatively more capable of limiting the spread of influenza virus in cell culture than are glass-adhering cells. These results suggest that mice immunized with influenza virus have sensitized spleen cells that elaborate antibody to influenza virus, which can act locally to control the spread of infection.

Early detection of influenza virus in cell culture by means of immunofluorescence.

An indirect fluorescent antibody technique using human serum for the rapid detection of influenza A virus in monkey kidney cell cultures is described. Influenza A2 virus was isolated from 38 of 88 nose/throat swabs received from patients with suspected influenza during the winter of 1971-72.Thirty-two strains isolated in monkey kidney cell cultures were identified by immunofluorescence on the day haemadsorption was observed, 22 of them within a week of receipt of the specimen. Human antiserum was found to be very satisfactory for this purpose.