scholarly journals STUDIES ON PERSISTENT INFECTIONS OF TISSUE CULTURES

1958 ◽  
Vol 108 (4) ◽  
pp. 537-560 ◽  
Author(s):  
Gertrude Henle ◽  
Friedrich Deinhardt ◽  
Victor V. Bergs ◽  
Werner Henle
Keyword(s):  
Chick Embryo ◽  
Influenza A ◽  
Aerobic Glycolysis ◽  
Horse Serum ◽  
Cellular Growth ◽  
Prolonged Treatment ◽  
Plaque Morphology ◽  
Persistently Infected ◽  
Persistent Infections ◽  
The Media

Inoculation of the MCN and Lung-To lines of human cells in continuous culture with Newcastle disease (NDV), mumps, or 6-6 viruses led to slight cytopathic effects (CPE) if the multiplicity of infection exceeded one. On second passage or with smaller initial inocula no CPE became apparent. The viruses multiplied, however, as determined by titrations in HeLa cultures or chick embryos. Indeed, persistently infected sublines of MCN and Lung-To were readily established without resort to special manipulations and some of these have been carried now for over 18 months on the same media and schedules as the uninfected parent strains. The viruses were found to be associated mainly with the cells and only 1, or at most 10 per cent of it was detectable in the media. The titers obtained were always low in relation to the available cell population. Reduction or even omission of the horse serum component in the media or ultraviolet irradiation of the cultures did not increase the yield of virus, and CPE became apparent only when similarly treated, uninfected cultures were, likewise, affected by the manipulations. The persistently infected cultures differed from their uninfected counterparts in that they exhibited (a) decreased cellular growth rates and ultimate yields; (b) increased aerobic glycolysis; and (c) a high degree of resistance to cytopathogenic viruses, influenza A (PR8), herpes simplex and, especially vesicular stomatitis (VSV) viruses. Prolonged treatment of persistently infected cultures by addition of specific antiviral immune sera to the media reduced significantly the amount of virus present and the degree of resistance to VSV. However, upon removal of the sera after as many as 187 days of treatment the viruses reappeared in all but one instance. The cured culture, on reinfection, became again persistently infected. No evidence was obtained to indicate genetic inhomogeneity of the cell populations. Of 50 cloned MCN lines none was destroyed by NDV and all became persistently infected. None were initially resistant to VSV but all after establishment of persistent NBV infection. All 39 cloned lines derived from MCNNDV cultures in the presence of anti-NDV serum, were free of virus and susceptible to VSV, and all acquired persistent infections and with it resistance to VSV following inoculation of NDV. NDV maintained in MCN cultures differed from the parent, chick embryo-adapted strain with respect to its plaque morphology. Whereas the former yielded only plaques on monolayers of chick embryo fibroblasts which were of pin-point size and hazy, those obtained with the latter were rarely of this type and mostly large and clear. This apparent selection of virus particles did not alter significantly their behavior with respect to cytopathogenicity for uninfected MCN cultures.

scholarly journals STUDIES ON PERSISTENT INFECTIONS OF TISSUE CULTURES

1959 ◽  
Vol 110 (4) ◽  
pp. 525-541 ◽  
Author(s):  
Werner Henle ◽  
Gertrude Henle ◽  
Friedrich Deinhardt ◽  
Victor V. Bergs
Keyword(s):  
Hela Cells ◽  
Persistent Infection ◽  
Influenza A ◽  
High Speed ◽  
Decisive Role ◽  
Persistent Infections ◽  
Interference Test ◽  
The Media ◽  
Vesicular Stomatitis ◽  
Reproductive Cycles

In previous reports of this series, it was shown that persistent infection of MCN cultures with certain myxoviruses rendered the cells insusceptible to superinfection by several cytopathogenic viruses. It was thought that production of an interferon might be the cause of this resistance and efforts to confirm this suggestion have been presented. Addition of ultraviolet-inactivated myxoviruses (mumps, Newcastle disease, influenza A, and Sendai) to MCN cultures for periods of 2 to 3 hours, followed by washing and refeeding of the cells, led to the subsequent release into the media of a substance which induced in fresh MCN cells a transitory resistance to infection by vesicular stomatitis virus, and prevented incomplete reproductive cycles of influenza A and Sendai viruses. Media containing this substance were free of detectable hemagglutinating activity and viral complement-fixing antigens. The substance was not neutralized by specific antiviral sera; it was not sedimentable by high speed centrifugation; it was not adsorbed onto red cells; but it was inactivated by trypsin. Thus, its properties matched those of the interferon described by Isaacs and his associates. A comparison of the extent of resistance induced in MCN cells by decreasing doses of ultraviolet-inactivated myxoviruses (interference test) and the protection afforded by the media removed from the cultures prior to challenge and transferred to fresh MCN tubes (interferon test) revealed that wherever interference became detectable in the cells, the media of the corresponding cultures contained some interferon. Interferon was obtained by inactivated myxoviruses also from primary cell cultures by the same techniques, but not from HeLa cells. Interferons derived from one type of culture may protect others equally well or show a certain degree of host specificity in that resistance in homologous cells may be somewhat more pronounced than in heterologous cultures. No resistance could be induced in HeLa cells by the interferon preparations employed. Interferon was detected also in MCN cultures, persistently infected with mumps virus. Its concentration was apparently too small in carrier cultures maintained as routine to be measurable. However, when the cells were grown in heavy sheets in roller bottles, and especially when the volume of medium was reduced for several days prior to harvest, interferon became readily detectable. These results strengthen the suggestion that interferon may play a decisive role in the establishment and maintenance of persistent infections in the system under study. Its nature, source, mode of action, and exact role in persistent infection remains to be elucidated.

scholarly journals STUDIES ON INFLUENZA VIRUS

1941 ◽  
Vol 73 (5) ◽  
pp. 581-599 ◽  
Author(s):  
Edwin H. Lennette ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Chick Embryo ◽  
Influenza A Virus ◽  
Influenza A ◽  
Complement Fixation ◽  
Soluble Antigen ◽  
Swine Virus ◽  
Specific Fixation ◽  
Mouse Lungs

Influenza complement fixation tests designed for use with ferret serum are described. Complement-fixing antigens derived from influenza ferret lungs were unsatisfactory due to their low content of soluble antigen; those prepared from mouse lungs or developing chick embryo membranes proved to be better antigenically and were reliable when the various reagents in the test were properly adjusted to eliminate non-specific fixation of complement. The results of cross complement fixation tests indicated that the soluble antigens of the PR8 and W.S. strains of influenza A virus were closely similar, if not identical. They indicated also that the soluble antigen of swine virus possessed components present in the antigens of the human strains of virus.

scholarly journals Type I Interferon acts as a major barrier to the establishment of infectious bursal disease virus (IBDV) persistent infections

2020 ◽  
pp. JVI.02017-20
Author(s):  
Laura Broto ◽  
Nicolás Romero ◽  
Fernando Méndez ◽  
Elisabet Diaz-Beneitez ◽  
Oscar Candelas-Rivera ◽  
...  
Keyword(s):  
Disease Virus ◽  
Cell Lines ◽  
Infectious Bursal Disease Virus ◽  
Infectious Bursal Disease ◽  
Type I ◽  
Persistently Infected ◽  
Persistent Infections ◽  
Avian Pathogen ◽  
Bursal Disease ◽  
Infected Cells

Infectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is a highly relevant avian pathogen causing both acute and persistent infections in different avian hosts. Here, we describe the establishment of clonal, long-term, productive persistent IBDV infections in DF-1 chicken embryonic fibroblasts. Although virus yields in persistently-infected cells are exceedingly lower than those detected in acutely infected cells, the replication fitness of viruses isolated from persistently-infected cells is higher than that of the parental virus. Persistently-infected DF-1 and IBDV-cured cell lines derived from them do not respond to type I interferon (IFN). High-throughput genome sequencing revealed that this defect is due to mutations affecting the IFNα/β receptor subunit 2 (IFNAR2) gene resulting in the expression of IFNAR2 polypeptides harbouring large C-terminal deletions that abolish the signalling capacity of IFNα/β receptor complex. Ectopic expression of a recombinant chicken IFNAR2 gene efficiently rescues IFNα responsiveness. IBDV-cured cell lines derived from persistently infected cells exhibit a drastically enhanced susceptibility to establishing new persistent IBDV infections. Additionally, experiments carried out with human HeLa cells lacking the IFNAR2 gene fully recapitulate results obtained with DF-1 cells, exhibiting a highly enhanced capacity to both survive the acute IBDV infection phase and to support the establishment of persistent IBDV infections. Results presented here show that the inactivation of the JAK-STAT signalling pathway significantly reduces the apoptotic response induced by the infection, hence facilitating the establishment and maintenance of IBDV persistent infections.IMPORTANCE Members of the Birnaviridae family, including infectious bursal disease virus (IBDV), exhibit a dual behaviour, causing acute infections that are often followed by the establishment of life-long persistent asymptomatic infections. Indeed, persistently infected specimens might act as efficient virus reservoirs, hence potentially contributing to virus dissemination. Despite the key importance of this biological trait, information about mechanisms triggering IBDV persistency is negligible. Our report evidences the capacity of IBDV, a highly relevant avian pathogen, to establishing long-term, productive, persistent infections in both avian and human cell lines. Data presented here provide novel and direct evidence about the crucial role of type I IFNs on the fate of IBDV-infected cells and their contribution to controlling the establishment of IBDV persistent infections. The use of cell lines unable to respond to type I IFNs opens a promising venue to unveiling additional factors contributing to IBDV persistency.

scholarly journals Type I Interferon acts as a major barrier to the establishment of infectious bursal disease virus (IBDV) persistent infections

2020 ◽  
Author(s):  
Laura Broto ◽  
Nicolás Romero ◽  
Fernando Méndez ◽  
Elisabet Diaz-Beneitez ◽  
Oscar Candelas-Rivera ◽  
...  
Keyword(s):  
Disease Virus ◽  
Cell Lines ◽  
Infectious Bursal Disease Virus ◽  
Infectious Bursal Disease ◽  
Type I ◽  
Persistently Infected ◽  
Persistent Infections ◽  
Avian Pathogen ◽  
Bursal Disease ◽  
Infected Cells

ABSTRACTInfectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is a highly relevant avian pathogen causing both acute and persistent infections in different avian hosts. Here, we describe the establishment of clonal, long-term, productive persistent IBDV infections in DF-1 chicken embryonic fibroblasts. Although virus yields in persistently-infected cells are exceedingly lower than those detected in acutely infected cells, the replication fitness of viruses isolated from persistently-infected cells is higher than that of the parental virus. Persistently-infected DF-1 and IBDV-cured cell lines derived from them do not respond to type I interferon (IFN). High-throughput genome sequencing revealed that this defect is due to mutations affecting the IFNα/β receptor subunit 2 (IFNAR2) gene resulting in the expression of IFNAR2 polypeptides harbouring large C-terminal deletions that abolish the signalling capacity of IFNα/β receptor complex. Ectopic expression of a recombinant chicken IFNAR2 gene efficiently rescues IFNα responsiveness. IBDV-cured cell lines derived from persistently infected cells exhibit a drastically enhanced proneness to establishing new persistent IBDV infections. Additionally, experiments carried out with human HeLa cells lacking the IFNAR2 gene fully recapitulate results obtained with DF-1 cells, exhibiting a highly enhanced capacity to both survive the acute IBDV infection phase and to support the establishment of persistent IBDV infections. Results presented here show that the inactivation of the JAK-STAT signalling pathway significantly reduces the apoptotic response induced by the infection, hence facilitating the establishment and maintenance of IBDV persistent infections.IMPORTANCEMembers of the Birnaviridae family, including infectious bursal disease virus (IBDV), exhibit a dual behaviour, causing acute infections that are often followed by the establishment of life-long persistent asymptomatic infections. Indeed, persistently infected specimens might act as efficient virus reservoirs, hence potentially contributing to virus dissemination. Despite the key importance of this biological trait, information about mechanisms triggering IBDV persistency is negligible. Our report evidences the capacity of IBDV, a highly relevant avian pathogen, to establishing long-term, productive, persistent infections in both avian and human cell lines. Data presented here provide novel and direct evidence about the crucial role of type I IFNs on the fate of IBDV-infected cells and their contribution to controlling the establishment of IBDV persistent infections. The use of cell lines unable to respond to type I IFNs opens a promising venue to unveiling additional factors contributing to IBDV persistency.

Persistent Infection of Mouse Fibroblasts (L Cells) with Chlamydia psittaci : Evidence for a Cryptic Chlamydial Form

1980 ◽  
Vol 30 (3) ◽  
pp. 874-883
Author(s):  
James W. Moulder ◽  
Nancy J. Levy ◽  
Laura P. Schulman
Keyword(s):  
Host Cell ◽  
Chlamydia Psittaci ◽  
Host Cells ◽  
Developmental Cycle ◽  
Persistently Infected ◽  
Mouse Fibroblasts ◽  
Persistent Infections ◽  
L Cells ◽  
Infected Cells ◽  
Parasite Multiplication

When monolayers of mouse fibroblasts (L cells) were infected with enough Chlamydia psittaci (strain 6BC) to destroy most of the host cells, 1 in every 10 5 to 10 6 originally infected cells gave rise to a colony of L cells persistently infected with strain 6BC. In these populations, the density of L cells and 6BC fluctuated periodically and reciprocally as periods of host cell increase were followed by periods of parasite multiplication. Successive cycles of L-cell and 6BC reproduction were sustained indefinitely by periodic transfer to fresh medium. Isolation of L cells and 6BC from persistent infections provided no evidence that there had been any selection of variants better suited for coexistence. Persistently infected populations consisting mainly of inclusion-free L cells yielded only persistently infected clones, grew more slowly, and cloned less efficiently. They were also almost completely resistant to superinfection with high multiplicities of either 6BC or the lymphogranuloma venereum strain 440L of Chlamydia trachomatis . These properties of persistently infected L cells may be accounted for by assuming that all of the individuals in these populations are cryptically infected with 6BC and that cryptic infection slows the growth of the host cell and makes it immune to infection with exogenous chlamydiae. According to this hypothesis, the fluctuations in host and parasite density occur because some factor periodically sets off the conversion of cryptic chlamydial forms into reticulate bodies that multiply and differentiate into infectious elementary bodies in a conventional chlamydial developmental cycle.

scholarly journals Baloxavir Marboxil Treatment of Nude Mice Infected With Influenza A Virus

2019 ◽  
Vol 221 (10) ◽  
pp. 1699-1702 ◽  
Author(s):  
Maki Kiso ◽  
Seiya Yamayoshi ◽  
Jurika Murakami ◽  
Yoshihiro Kawaoka
Keyword(s):  
Nude Mice ◽  
Influenza A ◽  
Body Weight Loss ◽  
Resistant Mutant ◽  
Prolonged Treatment ◽  
Drug Resistant ◽  
Neuraminidase Inhibitors ◽  
Drug Resistant Mutant ◽  
Suboptimal Dose ◽  
Resistant Mutants

Abstract Background Immunocompromised patients infected with influenza virus require prolonged treatment with neuraminidase inhibitors, because these patients are not able to eradicate the virus from the respiratory tract, leading to the emergence of drug-resistant mutant viruses. Methods In this study, we examined the efficacy of baloxavir marboxil in nude mice that were immunologically deficient. Results Daily treatment with a suboptimal dose of baloxavir marboxil increased the survival time of the virus-infected nude mice but did not clear the virus from their respiratory organs, resulting in gradual body weight loss after termination of treatment. Conclusions Despite the prolonged baloxavir marboxil treatment, few resistant mutants were detected.

scholarly journals Effect of Prolonged Treatment with Azithromycin, Clarithromycin, or Levofloxacin on Chlamydia pneumoniae in a Continuous-Infection Model

2002 ◽  
Vol 46 (2) ◽  
pp. 409-412 ◽  
Author(s):  
Andrei Kutlin ◽  
Patricia M. Roblin ◽  
Margaret R. Hammerschlag
Keyword(s):  
Chlamydia Pneumoniae ◽  
Day Treatment ◽  
Prolonged Treatment ◽  
Infection Model ◽  
Necrosis Factor Alpha ◽  
Persistent Infections ◽  
Cytokine Levels ◽  
Factor Alpha ◽  
Vitro Model

ABSTRACT Persistent infections with Chlamydia pneumoniae have been implicated in the development of chronic diseases, such as atherosclerosis and asthma. Although azithromycin, clarithromycin, and levofloxacin are frequently used for the treatment of respiratory C. pneumoniae infections, little is known about the dose and duration of therapy needed to treat a putative chronic C. pneumoniae infection. In this study, we investigated the effect of prolonged treatment with azithromycin, clarithromycin, or levofloxacin on the viability of C. pneumoniae and cytokine production in an in vitro model of continuous infection. We found that a 30-day treatment with azithromycin, clarithromycin, and levofloxacin at concentrations comparable to those achieved in the pulmonary epithelial lining fluid reduced but did not eliminate C. pneumoniae in continuously infected HEp-2 cells. All three antibiotics decreased levels of interleukin-6 (IL-6) and IL-8 in HEp-2 cells, but this effect appeared to be secondary to the antichlamydial activity, as the cytokine levels correlated with the concentrations of microorganisms. The levels of IL-1β, IL-4, IL-10, tumor necrosis factor alpha, and gamma interferon were too low to assess the effect of antibiotics. These data suggest that the dosage and duration of antibiotic therapy currently being used may not be sufficient to eradicate a putative chronic C. pneumoniae infection.

scholarly journals In Vitro Combinations of Baloxavir Acid and Other Inhibitors against Seasonal Influenza A Viruses

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1139
Author(s):  
Liva Checkmahomed ◽  
Blandine Padey ◽  
Andrés Pizzorno ◽  
Olivier Terrier ◽  
Manuel Rosa-Calatrava ◽  
...  
Keyword(s):  
Cell Viability ◽  
Influenza A ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Prolonged Treatment ◽  
Influenza A Viruses ◽  
Human Airway Epithelium ◽  
Influenza A H1n1 ◽  
Approved Drugs

Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors (baloxavir marboxil and favipiravir) can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations.

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