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1.
Simian virus 40 integration sites in the genome of virus-transformed mouse cells. 总被引:3,自引:3,他引:0
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To gain information on the specificity of simian virus 40 (SV40) integration in the genome of transformed cells, mouse 3T3 cells were transformed by a temperature-sensitive (ts) SV40 mutant, using high multiplicity of infection (MOI). Transformed cells were superinfected with wild-type (wt) virus at high MOI. Clones were isolated and fused with permissive BSC-1 cells to promote virus rescue. All rescued viruses were of the ts type only. When the high-MOI transformants were infected with 3H-labeled wt SV40, the amount of radioactivity associated with their nuclear fraction was found to be similar to that of 3T3 cells. 3T3 cells were then transformed by ts SV40 at low MOI and superinfected by wt virus at high MOI. Upon fusion with BSC-1 cells, most clones produced both ts and wt virus. These results suggest that the number of stable SV40 integration sites in the 3T3 genome is limited, since they can be saturated by transformation at high MOI. When the MOI is low, the sites are not saturated and a subsequent infection can lead to integration. 相似文献
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State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules. 总被引:40,自引:22,他引:18
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The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives. 相似文献
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Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses. 相似文献
6.
Homologous interference of lymphocytic choriomeningitis virus involves a ribavirin-susceptible block in virus replication. 总被引:1,自引:1,他引:0
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Depending on the multiplicity of infection (MOI), infection of L929 cells results in either productive lymphocytic choriomeningitis virus replication or homologous interference M. Bruns, A. Gessner, H. Lother, and F. Lehmann-Grube, Virology 166:133-139, 1988). As shown in this communication, productive lymphocytic choriomeningitis virus replication as observed at a low MOI was effectively inhibited by ribavirin. In contrast, virus yields increased if cells were infected with a high MOI and in the presence of 5 microM of the antiviral compound. This drug-dependent release of infectious virus was preceded by enhanced nucleoprotein (NP) synthesis, a change in intracellular NP distribution, and by an onset of glycoprotein synthesis. It is therefore proposed that this block in viral replication is brought about by a posttranslational effect on a viral gene product, probably the NP, present in reasonably large quantities both during homologous interference as well as persistent infection. 相似文献
7.
Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues 总被引:1,自引:0,他引:1
Valeria M. Rodas Fabiano H. Marques Marcelo T. Honda Daniela M. Soares Soraia A. C. Jorge Marta M. Antoniazzi Claudia Medugno Maria E. B. Castro Bergmann M. Ribeiro Marlinda L. Souza Aldo Tonso Carlos A. Pereira 《Cytotechnology》2005,48(1-3):27-39
We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors.
We have assayed the kLa of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients
consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than
in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs
production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a
cell concentration at infection (CCI) of 1 to 2.5×106 cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed
to be dependent on the MOI used, and the μX at the exponential growth phase in infected and non-infected cultures were, respectively,
of 0.2832 and 0.3914 (day−1). The glucose consumption and lactate production were higher in the infected cultures (μGlucose and μLactate of, respectively,
0.0248 and 0.0089×10−8 g/cell×day in infected cultures and 0.0151 and 0.0046×10−8 g/cell×day in non infected ones). The glutamine consumption did not differ in both cultures (μGlutamine of 0.0034 and 0.0037×10−8 g/cell×day in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a
higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these
cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective
decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance
of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 106 to 2×106 cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high
MOI showed comparable DL50. Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better
environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB
virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase
in the DL50. The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered
DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy
(TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described
here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control. 相似文献
8.
Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system. 相似文献
9.
The possibility that virus multiplication may be inhibited by interaction of infected cells with uninfected cells was tested by experiments, using human adenovirus type 12 (Ad 12). Permissive human cells (human embryonic kidney = HEK, KB or HeLa) were infected and seeded on uninfected or infected “nonpermissive” cell (human embryonic lung = HEL) monolayers, and virus yields or proportions of viral antigen-synthesizing cells were compared with each other. Both the virus yields and the proportions of viral antigen-positive cells were not reduced significantly when seeded on infected HEL cells, while when seeded on uninfected HEL cells both of them were reduced remarkably, compared with the yield and the proportion of controls seeded on glass. Similar results were obtained regardless of the type of permissive cells, HEK, KB, or HeLa. Similar reduction of the yield was observed when seeded on HEL cells infected with Ad 12 inactivated by heat or by antiserum, and partial reduction was observed when seeded on HEL cells infected with UV-inactivated Ad 12, depending on the extent of UV dosis. These experiments showed that intracellular virus multiplication may be inhibited by interaction of infected cells with uninfected cells, and this may be due to the difference in the cell surface structure. 相似文献
10.
Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.
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Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion. 相似文献
11.
Replication of Epstein-Barr virus: ultrastructural and immunofluorescent studies of P3HR1-superinfected Raji cells.
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We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (less than or equal to 50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (greater than or equal to 300 particles per cell), a productive cycle was the rule. 相似文献
12.
The human cytomegalovirus major immediate-early distal enhancer region is required for efficient viral replication and immediate-early gene expression
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The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI. 相似文献
13.
Acquisition of Sequences Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid 总被引:59,自引:36,他引:23
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The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA. 相似文献
14.
Background
Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis.Results
The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results.Conclusions
(i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development. 相似文献15.
Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers 总被引:2,自引:0,他引:2
Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qbeta) were exposed to UV radiation from 0 to 150 mJ.cm-2. PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qbeta having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ.cm-2. In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ.cm-2 and 60 mJ.cm-2, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation. 相似文献
16.
We have tested the hypothesis that some transformation-defective (td) viruses grow faster than the avian sarcoma viruses (ASV) from which they are derived, resulting in establishment of interference by the td virus and suppression of the ASV multiplication. Using an ASV of subgroup A (ASV-A) that does not contain td virus and an independently isolated tdASV-A, we performed separate and mixed infections to test this hypothesis. At multiplicities of 1 or less, tdASV alone grew to higher titers and more rapidly than ASV alone. In mixed infections at low multiplicities that allowed spread of progeny virus, when as little as 10% of the virus inoculum was td virus, there was an excess of td virus by 2 days after infection and a decrease in the titer of ASV relative to a control infection with no td virus. In mixed infections at high multiplicities which minimized spread of progeny virus, there was no excess of td virus and the titer of ASV was not decreased relative to the control infection with no td virus. These data support the hypothesis that we proposed and indicate that deletions in the ASV src gene may not be a high-frequency event. We also present data concerning the amounts of unintegrated viral DNA found after the separare and mixed infections. There was no simple correlation between the amounts of unintegrated viral DNA early after infection and the titers of virus produced, indicating perhaps that virus production was determined by integrated viral DNA. 相似文献
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Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells. 总被引:1,自引:1,他引:0
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Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells. 相似文献
20.
Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein
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Kearney K Chen D Taraporewala ZF Vende P Hoshino Y Tortorici MA Barro M Patton JT 《The EMBO journal》2004,23(20):4072-4081
Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3'-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0-5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3'CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines. 相似文献