Total Virus Production Research Articles

Quorum sensing signal disrupts viral infection dynamics in the coccolithophore Emiliania huxleyi

Viruses that infect phytoplankton are abundant in all regions of the global ocean. Despite their ubiquity, little is understood regarding how biotic interactions can alter virus infection success as well as the fate of phytoplankton hosts. In previous work, the bacterially derived compound 2-heptyl-4-quinolone (HHQ) has been shown to protect the cosmopolitan coccolithophore Emiliania huxleyi from virus-induced mortality. The present study explores the potential mechanisms through which protection is conferred. Using a suite of transmission electron microscopy and physiological diagnostic staining techniques, we show that when E. huxleyi is exposed to HHQ, viruses can gain entry into cells but viral replication and release is inhibited. These findings are supported by a smaller burst size, as well as lower infectious and total virus production when the host is treated with nanomolar concentrations of HHQ. Additionally, diagnostic staining results indicate that programmed cell death markers commonly associated with viral infection are not activated when infected E. huxleyi are exposed to HHQ. Together, these results suggest that the ability of HHQ to inhibit infectious viral production protects the alga not from getting infected, but from cell lysis. This work identifies a new mechanistic role of bacterial quorum sensing molecules in mediating viral infections in marine microbial systems.

Antiherpes simplex virus type 2 activity of the antimicrobial peptide subtilosin.

In this study, we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV-2) in cell cultures and we investigated subtilosin mode of action. We determined, using a virus yield inhibition assay, that noncytotoxic concentrations of subtilosin inhibit HSV-2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h postinfection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin-treated culture. On the other hand, at high concentrations, subtilosin displays virucidal action. Subtilosin displays antiviral and virucidal actions against HSV-2. The target of subtilosin inhibitory effect would be late stages of the viral replicative cycle such as viral glycoprotein intracellular transport. Given its antimicrobial activity and its safety for human tissues, subtilosin could represent a valuable alternative to be considered in the development of new microbicide formulations.

Syncytia formation affects the yield and cytotoxicity of an adenovirus expressing a fusogenic glycoprotein at a late stage of replication

Fusogenic membrane glycoproteins (FMGs) may enhance the cytotoxicity of conditionally replicative adenoviruses. However, expression at early stages of infection impairs virus replication. We have inserted the hyperfusogenic form of the gibbon ape leukemia virus (GALV) envelope glycoprotein as a new splice unit of the major late promoter (MLP) to generate a replication-competent adenovirus expressing this protein. At high multiplicity of infection (MOI), this virus replicated efficiently forming clumps of fused cells and showing a faster release. In contrast, at low MOI, infected cells formed syncytia where only one nucleus contained virus DNA, decreasing total virus production but increasing cytotoxicity.

Susceptibility of Human Corneal Endothelial Cells to HSV-1 Infection

Purpose: The purpose of this study was to examine the intrinsic susceptibility of cultured human corneal endothelial cells (HCEC) to herpes simplex virus-1 (HSV-1) infection. We compared HSV-1 adsorption, kinetics of HSV-1 production, and pattern of viral plaque formation in cultured HCEC with those of a cell line used routinely for laboratory HSV propagation (African green monkey kidney fibroblast CV-1 cells). Methods: Cultured HCEC and CV-1 cells were exposed to the McKrae strain of HSV-1 at 5 and 0.0001 multiplicities of infection (MOI). Using the 50% tissue culture infectious dose (TCID50) titration method, viral adsorption (at 5 MOI) and total virus production (at 5 and 0.0001 MOI) were compared to assess both susceptibility to viral attachment and productive viral infection, respectively. Additionally, visual observations were made at 0.0001 MOI using bright-field microscopy and immunofluorescence staining of viral antigens to compare patterns of viral spread in confluent monolayers of both cell types. Results: The percentage of HSV-1 virion particles adsorbed by cultured HCEC and CV-1 cells was similar (35.9% and 33.0%, respectively, p = 0.07, NS), indicating similar susceptibility of the two cell types to initial HSV-1 attachment and adsorption. However, maximum total virus production was more than 3-fold higher for HCEC than for CV-1 cells (p < 0.005), suggesting higher susceptibility of HCEC cells to productive viral infection. Immunofluorescence studies of infected cell monolayers corroborated these quantitative findings, with HCEC monolayers demonstrating more rapid progression of cytopathic effect than CV-1 monolayers. Conclusions: In comparison to reference CV-1 cells, cultured HCEC show similar susceptibility to HSV-1 adsorption, but higher capacity to support productive HSV-1 infection. Our results suggest that human corneal endothelial cells may be inherently susceptible to HSV-1 infection.

Replication and Tissue Tropism of the Alphavirus Sindbis in the Mosquito Aedes albopictus

We have examined the replication and tissue distribution of the alphavirus Sindbis in the mosquito Aedes albopictus. Parenteral inoculation of virus resulted in an acute infection accompanied by rapid virus replication and a persistent infection, during which total virus production was reduced. Acute and persistent phase virus RNA synthesis, virus production, and organ-specific distribution of infection were determined over an 18-day incubation period. Organs were classified as refractory (ovarioles, Malpighian tubules), cleared (head ganglia), transient (salivary glands, anterior midgut, posterior midgut, and thoracic muscle), or persistent (fat bodies-hemolymph, hindgut, and tracheole-associated cells) according to the onset, peak, and duration of Sindbis virus antigens within that particular organ. Virus was identified in respiratory tissue by immunological and ultrastructural methods. This represents a novel tropism for an alphavirus. These findings demonstrate that the cells of mature insect organs respond differently to virus infection. Correlations among virus replication, virus RNA synthesis, and organ-specific clearing of a pantropic infection were observed. We suggest that the underlying temporal and spatial kinetics that characterize this virus-invertebrate interaction may reflect a mechanism for the modulation of the arbovirus titer seen in the mosquito host.

Comparative Replication, Mortality, and Inclusion Body Production of the Autographa californica1 Nuclear Polyhedrosis Virus in Heliothis sp.12

The histopathology was studied and mortality, polyhedra production, total virus production, and nonoccluded virus production were measured in Heliothis zea (Boddie) and H. virescens (F.) (Lepidoptera: Noctuidae) larvae infected with the nuclear polyhedrosis virus isolated from Autographa californica (Speyer) (Lepidoptera: Noctuidae). Both per os and injection routes of inoculation were used, and most tests were purposely biased toward demonstrating viral replication in H. zea. In addition to the great difference in the histopathological responses of the two hosts, large differences in susceptibility via both routes of inoculation were observed. Polyhedra formation, total virus production, and nonoccluded virus production were severely reduced in H. zea as compared with H. virescens larvae. In some cases, these differences exceeded 5,992-fold, even with the tests biased toward obtaining increased responses from H. zea larvae.

The localization of influenza virus in the respiratory tract of ferrets: susceptible nasal mucosa cells produce and release more virus than susceptible lung cells.

Infectious virus production by ferret nasal mucosa and lung organ cultures has been monitored in both tissue pieces and medium over 24 h following inoculation with an Asian (H2N2) strain of influenza virus. Freshly prepared cultures of nasal mucosa produced approx. 10-fold more virus per cell than fresh lung cultures. Also the nasal mucosa cells liberated into the medium a greater proportion (mean 31%) of the total virus produced than did fresh lung (mean 6%). Maintenance of lung explants for 24 h prior to inoculation resulted in a 20- to 100-fold increase in the amount of virus released. However, total virus production by fresh and maintained lung was similar. Trypsin did not increase the infectivity of virus released from any of the cultures, indicating that the haemagglutinin in the virus particles was cleaved. Similar results were obtained with a Hong Kong (H3N2) virus strain. Hence, one factor operating in the lower susceptibility of the lung compared with the nasal mucosa in vivo may be a lower capacity of lung cells both to produce and release influenza virus.

A mathematical analysis of concomitant virus replication and heat inactivation.

A mathematical analysis of virus production with accompanying heat inactivation, from which the rate of virus release and total virus production are readily calculated, is presented. Applications of this analysis for Sindbis and Chikungunya viruses are discussed.

Cell-virus interactions with the polyoma virus: I. Studies on the lytic interaction in the mouse embryo system

A study of the lytic cycle of polyoma virus multiplication on mouse embryo monolayer cultures has shown that the growth curve characteristics are similar to some cell-killing viruses that do not produce tumors. In single-cycle growth experiments, progeny virus was first detected at 22 and 24 hours after infection as increases in total and free virus, respectively. After 33 hours, the increase in total virus leveled off to a plateau. The presence of viral antigen in the nuclei of infected cells, as demonstrated by fluorescent antibody staining, was first detected at the twentieth hour after infection. Following this, the proportion of cells with fluorescent viral antigen increased gradually during this single cycle of growth, thus showing the asynchrony of polyoma virus production by the cells in this system. It was shown that a large fraction of the progeny virus produced remains cell associated and that the relationship between free virus and total virus can be influenced by the concentration of horse serum in the culture medium. Although total virus production was not decreased when higher horse serum levels were incorporated into the culture medium, the fraction of total virus represented by free virus was significantly reduced. In order to convert approximately 90 % of the cells to virus production, it was found necessary to inoculate the culture with a large excess of virus (virus:cell ratio, 940:1). In cultures inoculated at virus:cell ratios of 533:1 and less, only 10 % of the cells, as measured by both infective center assay and fluorescent antibody staining, were detected as virus producers. Based upon the proportion of cells detected as virus producers, the average yields of virus were 800–1000 PFU per cell. The characteristics of the plaque assay used in these investigations, and which is based upon the lytic cycle of multiplication, have been further analyzed. In a study on the high thermostability of polyoma virus, the half-life at 37° C was found to be 7 days.