Intracellular Virus Titers Research Articles

Modulation of Zika virus replication via glycosphingolipids

The enveloped positive-sense RNA viruses including Zika virus (ZIKV) need host lipids to successfully replicate. The nature of the lipids and the replication step(s) where lipids are utilized often vary amongst viruses. In this study, we demonstrate that ZIKV particle envelope is significantly enriched in distinct sphingolipid species. To determine the role of sphingolipids in ZIKV replication, we leveraged a panel of sphingolipid-deficient cell lines. Notably, knockout of glucosylceramide and lactosylceramide synthase encoding genes (GCSKO; B4G5KO) resulted in a marked decrease in ZIKV titers. GCSKO or pharmacological inhibition of GCS also led to a significant decrease in ZIKV genome replication. Further analysis indicated that GCSKO reduced intracellular virus titers but had minimal impact on ZIKV binding. Restoration of B4G5 expression in B4G5KO cells or supplementing PDMP-treated cells with glucosylceramide led to a significant rescue of ZIKV replication. Altogether, our findings suggest that ZIKV needs glycosphingolipids to facilitate virus replication.

Viral replication and innate immunity of feline herpesvirus-1 virulence-associated genes in feline respiratory epithelial cells

Feline herpesvirus-1 (FHV-1) infection occurs worldwide and is a leading cause of respiratory and ocular diseases in cats. Current vaccines reduce the severity of symptoms but do not prevent infection and, therefore, do not provide defense against an establishment of latency and reactivation. We hypothesize that immunomodulation of FHV-1 is the cause of lack in protection and that deletion of virulence/immune modulatory genes of FHV-1 will enhance safety and immunogenicity. Our objective was to use feline respiratory epithelial cell (FREC) cultures to define in vitro growth characteristics and immunomodulation resulting from infection of FRECs with the virulent FHV-1 strain C27 (WT) and glycoprotein C-deletion (gC-), glycoprotein E-deletion (gE-), serine/threonine protein kinase-deletion (PK-), as well as gE and thymidine kinase-double-deletion (gE-TK-) mutants generated by bacterial artificial chromosome mutagenesis.Differentiated FRECs were mock inoculated or inoculated with WT, gC-, gE-, PK-, or gE-TK- mutants. Virus titration and real-time quantitative PCR assays were performed on samples collected at 1 hpi followed by 24 h intervals between 24 and 96 hpi to determine growth kinetics. Real-time PCR was used to quantitate IFNα, TNFα, IL-1β, IL-10, and TGFβ-specific mRNA levels. Immunoassays were performed to measure the protein levels of subsets of cytokines/chemokines secreted by FRECs.Inoculation of FRECs with gE-TK- resulted in significantly lower end-point titers than inoculation with WT or gE-. Both PK- and gC- inoculated FRECs also produced significantly lower end-point titers at 96 hpi than WT. Overall, intracellular virus titers were higher than those of extracellular virus. PCR results for viral DNA paralleled the virus titration results. Further, in contrast to WT inoculation, an increase in IFNα and IL-10 mRNA expression was not observed following inoculation with gE-TK- and PK-, but inoculation with gE-TK- and PK- did result in increased TGFβ expression in FRECs compared to responses following infection with WT. Moreover, gE-TK- and PK- blocked the inhibition of IL-8 and neutrophil chemoattractant (KC), which was observed following inoculation with WT.In summary, the results obtained in FRECs may be used to predict the safety and immunogenicity characteristics of these mutants in vivo. Our study highlights the value of the FREC system for studying replication kinetics/immune modulation factors of FHV-1 and screening prospective vaccine candidates before their use in experimental cats.

Coronaviruses induce entry-independent, continuous macropinocytosis.

ABSTRACTMacropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions.

Role of annexin A2 in the production of infectious hepatitis C virus particles.

Hepatitis C virus (HCV) is an important human pathogen affecting 170 million chronically infected individuals. In search for cellular proteins involved in HCV replication, we have developed a purification strategy for viral replication complexes and identified annexin A2 (ANXA2) as an associated host factor. ANXA2 colocalized with viral nonstructural proteins in cells harboring genotype 1 or 2 replicons as well as in infected cells. In contrast, we found no obvious colocalization of ANXA2 with replication sites of other positive-strand RNA viruses. The silencing of ANXA2 expression showed no effect on viral RNA replication but resulted in a significant reduction of extra- and intracellular virus titers. Therefore, it seems likely that ANXA2 plays a role in HCV assembly rather than in genome replication or virion release. Colocalization studies with individually expressed HCV nonstructural proteins indicated that NS5A specifically recruits ANXA2, probably by an indirect mechanism. By the deletion of individual NS5A subdomains, we identified domain III (DIII) as being responsible for ANXA2 recruitment. These data identify ANXA2 as a novel host factor contributing, with NS5A, to the formation of infectious HCV particles.

Effects of ozone exposure on inactivation of intra- and extracellular enterovirus 71

In this study, the potential of ozone in inactivating enterovirus 71 (EV71) free particles was investigated using either various ozone flow rates of 100, 80 or 60 mg/h or a constant flow rate of 80 mg/h, given to culture medium or various pH culture media containing EV71, respectively. Results demonstrated that EV71 inactivation by ozone was related to the kinetics of ozone solubility, ∼99% inactivation being obtained in the exponential phase of ozone solubility. However, the inactivation rate was dependent on the ozone input flow rate and positively enhanced at acidic pH. Inactivation of intracellular EV71 was also studied. At a constant ozone supply of 60 mg/h, a significant reduction of intracellular virus titer (≥99%, p < 0.01) was obtained after 45 or 60 min exposure but with low cell viability. Upon 30 min exposure, however, 45% cell viability was retained. The results indicate that the inactivating effect of ozone on intracellular EV71 virus is dependent on exposure duration.

Isolation of equine herpesvirus-1 lacking glycoprotein C from a dead neonatal foal in Japan

We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests in 5089 virions and 5089-infected cells, respectively. RT-PCR analysis revealed that the expression level of 5089 gC mRNA was reduced considerably compared to that of wild-type EHV-1. Sequencing analysis of the 5089 gC coding region showed a point mutation in the promoter region of the gC open reading frame. However, the mutation did not affect the promoter activity. These results suggested that the lack of gC in 5089 virions might be one of the reasons for spread of the virus by cell-to-cell infection and that gC mRNA expression might not be activated efficiently due to factors other than the mutation in the gC promoter region.

Replication of cytopathic and noncytopathic bovine viral diarrhea virus in zona-free and zona-intact in vitro-produced bovine embryos and the effect on embryo quality.

The aim of the present study was to determine whether or not cytopathic (CP) and noncytopathic (NCP) bovine viral diarrhea virus (BVDV) are able to replicate within in vitro-produced embryos and to investigate whether inoculation of embryos with BVDV affects their normal development. Zona pellucida (ZP)-free oocytes, zygotes, 8-cell-stage embryos, morulae, and hatched blastocysts (HB) were incubated for 1 h in 1 ml of Minimal Essential Medium containing 10(6.00) tissue culture infectious dose (TCID)50/ml NCP BVDV isolate 22,146 or 10(6.25) TCID50/ml CP BVDV strain Oregon C24V. At 0, 12, 24, 36, 48, 60, and 72 h postinoculation (hpi), groups of embryos were collected for virus titration. A small amount of newly produced virus was detected in 8-cell embryos at 60 hpi (10(1.8) TCID50/100 cells), but only for CP BVDV. For ZP-free morulae and HB, maximal intracellular virus titers were, respectively, 10(1.47) and 10(2.33) TCID50/100 cells at 48 hpi for the CP biotype and 10(0.64) and 10(0.84) TCID50/100 cells at 72 hpi for the NCP biotype. Only an infection with CP BVDV had a significant inhibitory effect on further development of ZP-free morulae. It can be concluded that ZP-free in vitro-produced embryos are permissive to an infection with BVDV, with increasing susceptibility of the embryos in accordance with their developmental stage. In contrast to observations in ZP-free in vitro-produced embryos, no virus replication or signs of embryonic degeneration were detected in ZP-intact in vitro-derived embryos.

Susceptibility of zona-intact and zona-free in vitro-produced bovine embryos at different stages of development to infection with bovine herpesvirus-1

The aim of the present study was to determine if BHV-1 is able to replicate within in vitro produced embryos and to investigate the degree to which the zona pellucida (ZP) is able to protect in vitro produced embryos against infection with BHV-1. Both ZP-intact and ZP-free matured oocytes, zygotes (1 d post insemination; 1dpi), 8-cell stage embryos (3 dpi), morulae (6 dpi) were incubated for 1 h in 1 ml of MEM containing 10 7.7 TCID 50/ml BHV-1 (Cooper strain). Three titers (10 5.7, 10 6.7 and 10 7.7 TCID 50/ml) of the Cooper strain were used for incubation of hatched blastocysts (9 dpi). Bovine embryonic lung cells (BEL) on microcarriers were inoculated following the same protocol as for the embryos. At 0, 12, 24, 36 and 48 h post inoculation (hpi), groups of embryos and BEL cells were collected for virus titration and for the determination of the percentage of viral antigen positive cells by immunofluorescence. For the 3 developmental stages in ZP-free embryos, similar maximal intracellular virus progeny titers were obtained at 24 to 48 hpi ranging from 10 1.32 to 10 1.43 TCID 50/ 100 embryonic cells. The intracellular virus titer in the BEL cells peaked at 10 3.08 TCID 50/ 100 BEL cells. The percentage of cells which expressed viral antigens was 13% in ZP-free hatched blastocysts, 17% in ZP-free morulae and 100% in BEL cells. In ZP-intact embryos, no replication of BHV-1 was detected. These results clearly show that only after removal of the zona pellucida, BHV-1 is able to replicate within the in vitro produced embryos, with only a subset of embryonic cells being fully susceptible.

Effect of dexamethasone on herpes simplex virus replication in mouse neuroblastoma cells (NB41 A3): Receptor characteristics

Monolayers of NB41A3 (MNB) cells were exposed to pharmacologic doses of dexamethasone (DXM). After 24-h, the cells were infected with herpes simplex virus type 1 (HSV-1) at a multiplicity of infection (m.o.i.) = 0.1. After every 24-h period, extracellular and intracellular virus aliquots were collected and frozen. The aliquots were titered in a standard plaque-forming assay. It was shown that the hormone led to a statistically significant increase of extra- and intracellular virus titers above the titers exhibited by these cells without added hormone. The same experiment was repeated in Vero cells, but the hormone did not elevate the resultant HSV-1 titers. A binding assay was performed on these two cell lines by use of 3H-DXM to determine if a DXM receptor was present. Specific binding was seen, but only in the MNB cell line. The Bmax of this receptor was 480 fmol/mg protein and it had a Kd of 2.3 nM. The S value of the receptor ligand complex equalled 8.0. These results indicate that cells possessing hormone receptors allow for a more efficient replication of the virus and suggest that these hormones may play an important role in the exacerbation of herpes simplex virus infection in vivo.

Adsorption, purification, and growth characteristics of hepatitis A virus strain HAS-15 propagated in fetal rhesus monkey kidney cells.

A human fecal isolate of hepatitis A virus strain HAS-15 was adapted to rapid growth in FRhK-4 cells by more than 20 7-day passages. A cell culture-derived inoculum of strain HAS-15 was used at a multiplicity of infection of 80 radioimmunofocus-forming units per cell, and a one-step growth curve was determined. Both intracellular production and supernatant release of infectious virions were evaluated. Detection of virus release into the medium directly corresponded to intracellular production of infectious virions. A classical eclipse period was not observed during the growth curve determinations; however, detectable infectious virion production was absent for approximately 20 h after infection. This 20-h period was immediately followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10(9) radioimmunofocus-forming units per ml was achieved, and this level remained essentially constant for up to 14 days after infection. The infectious virus and viral antigen produced during the growth cycle were ascertained by a radioimmunofocus assay and by a radioimmunoassay, respectively. Cell culture supernatants were negative for viral antigen as determined by the radioimmunoassay, even though as many as 10(8) hepatitis A virus radioimmunofocus-forming units per ml were found. An adsorption study was also performed with strain HAS-15 by using FRhK-4 cells. More than 99.9% of the infectious virus was adsorbed at 25 degrees C in less than 20 min.

Replication of Dengue Virus in Cultured Mosquito Cells at Suboptimal Temperature

AbstractCultured Aedes albopictus mosquito cells were infected at optimal (28°) and suboptimal (20°) temperatures with dengue virus type-3 at multiplicities of infection of 2.0 and 0.01. Extracellular and intracellular virus titers were determined over an interval of 6 weeks by fluorescent focus assay using hyperimmune mouse ascities fluid as a source of antibody. Upon infection at a multiplicity of 2.0, cultures at 28° reached peak virus titers in 3 days, while cultures at 20° required 14 days. At a lower infection multiplicity of 0.01, peak titers were reached in 5 days at 28°, but required 3 weeks at 20°. Viral titers maintained after the peak was reached were the same at both infection multiplicities and temperatures tested. At both 28° and 20° A. albopictus cultures became persistently infected with 20-30% of the cells containing immunofluorescent cell-associated viral antigen. These results indicate that the time required to reach peak infectious virus titers for dengue-3-infected A. albopictus cell...

Glucose consumption of cultured cells before and after infection with enteroviruses or virus nucleic acids

The sugar consumption of the same cell population was examined before and after infection with virulent or attenuated Type I Poliovirus, Mahoney and LSc strains or Echo 6 virus. The energy requirements of virus production, as derived from glucose are not different from that necessary for normal cell life, under the experimental conditions. There is no difference in this respect, if the infection starts with intact virus, or with viral RNA. Mahoney, LSc, Echo 6 viruses behaved alike. The titration of intracellular virus revealed the usual sequence of virus synthesis and definite intracellular virus titers at the end of 6 hours experimental period. Thus, the virus infection, adsorption, penetration, eclipse and re-synthesis of virus did not cause measurable quantitative difference from normal, regarding the glucose utilization, when the same cell population was assayed before and after inoculation with virus or viral nucleic acid.

Biochemical studies of meningopneumonitis virus multiplication in L cell suspension culture

As part of a general investigation of meningopneumonitis virus (MPV) multiplication in L cell, the nucleic acid content of the cells has been studied by direct chemical analysis at successive stage in the growth cycle.Cal 10 strain of MPV and L cell suspension culture were used. Measurements of virus adsorption rate, distribution of MPV after adsorption and infective center formation indicated that essentially all cells were infected within 1 hour after addition of virus at multiplicity more than 50 PFU per cell.Growth curves of MPV showed that an increase in intracellular virus began at 18.5 hours after infection and reached levels of 450-500 PFU per infected cell. It should be noted that the amount of intracellular virus titer during lag period dropped extremely below that number of infected cells, showing as low as 10-3 PFU per infected cell at the lowest point.In the experiments on the multiplication of infected cells, uninfected control cells multiplied until 25 hours, followed by stationary phase. But infected cell cultures did not show increase in cell number. Infected dead cells which were determined by staining with trypan blue were observed 20 hours after infection and almost all cells were dead within 35 hours after infection.Nitrogen content in uninfected cells was constant until 40 hours, but in infected cultures decline was observed subsequent after 25 hours of infection. Protein content was constant until 20 hours, followed by dropping down both in infected and uninfected cells.On the other hand, in analysing RNA and DNA contents per 106 cells, although they were constant during 40 hours in control cultures, significant increase was observed in infected cells. The RNA and DNA increment in infected cells was first detected after 10 and 15 hours of infection, respectively, which proceeded an increase of cell associated virus by 8.5 and 3.5 hours. RNA content reached maximum at 20 hours after infection, followed rapidly decreased. DNA content continued to increase and reached near maximum approximately 25 hours after infection. Maximum increment of RNA and DNA reached levels about 133% and 135% of control cells, respectively.From the results described above, it appears that both RNA and DNA are synthesized in MPV infected L cells. These polymer increment is demonstrable several hours before a significant increase in intracellular virus is found. The relationship of these increased nucleic acids to virus multiplication has been discussed.