Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

2008 ◽  
Vol 89 (5) ◽  
pp. 1220-1224 ◽  
Author(s):  
Mark P. Zwart ◽  
Eloy Erro ◽  
Monique M. van Oers ◽  
J. Arjan G. M. de Visser ◽  
Just M. Vlak
Keyword(s):  
Cell Culture ◽  
Purifying Selection ◽  
Serial Passage ◽  
Autographa Californica ◽  
Homologous Region ◽  
Deletion Mutants ◽  
Wild Type ◽  
Insect Larvae ◽  
Selection For

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective condition.

scholarly journals A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

2003 ◽  
Vol 69 (4) ◽  
pp. 2052-2057 ◽  
Author(s):  
James C. Bull ◽  
H. C. J. Godfray ◽  
David R. O'Reilly
Keyword(s):  
Cell Culture ◽  
Population Genetics ◽  
Trichoplusia Ni ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Cell Infection ◽  
Occlusion Bodies ◽  
Budded Virus

ABSTRACT Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

scholarly journals Recombinant Human Metapneumovirus Lacking the Small Hydrophobic SH and/or Attachment G Glycoprotein: Deletion of G Yields a Promising Vaccine Candidate

2004 ◽  
Vol 78 (23) ◽  
pp. 12877-12887 ◽  
Author(s):  
Stéphane Biacchesi ◽  
Mario H. Skiadopoulos ◽  
Lijuan Yang ◽  
Elaine W. Lamirande ◽  
Kim C. Tran ◽  
...  
Keyword(s):  
Cell Culture ◽  
Respiratory Tract ◽  
Human Metapneumovirus ◽  
Reverse Genetic System ◽  
Deletion Mutants ◽  
Upper Respiratory Tract ◽  
Respiratory Tract Disease ◽  
Wild Type ◽  
Tract Disease

ABSTRACT Human metapneumovirus (HMPV) has recently been identified as a significant cause of serious respiratory tract disease in humans. In particular, the emerging information on the contribution of HMPV to pediatric respiratory tract disease suggests that it will be important to develop a vaccine against this virus for use in conjunction with those being developed for human respiratory syncytial virus and the human parainfluenza viruses. A recently described reverse genetic system (S. Biacchesi, M. H. Skiadopoulos, K. C. Tran, B. R. Murphy, P. L. Collins, and U. J. Buchholz, Virology 321:247-259, 2004) was used to generate recombinant HMPVs (rHMPVs) that lack the G gene, the SH gene, or both. The ΔSH, ΔG, and ΔSH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Thus, the SH and G proteins are not essential for growth in cell culture. Apart from the absence of the deleted protein(s), the virions produced by the gene deletion mutants were similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. When administered intranasally to hamsters, the ΔG and ΔSH/G mutants replicated in both the upper and lower respiratory tracts, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were at least 40-fold and 600-fold restricted in replication in the lower and upper respiratory tract, respectively, compared to wild-type rHMPV. They also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Surprisingly, G is dispensable for protection, and the ΔG and ΔSH/G viruses represent promising vaccine candidates. In contrast, ΔSH replicated somewhat more efficiently in hamster lungs compared to wild-type rHMPV (20-fold increase on day 5 postinfection). This indicates that SH is completely dispensable in vivo and that its deletion does not confer an attenuating effect, at least in this rodent model.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k

2012 ◽  
Vol 93 (2) ◽  
pp. 389-399 ◽  
Author(s):  
Lopamudra Giri ◽  
Michael G. Feiss ◽  
Bryony C. Bonning ◽  
David W. Murhammer
Keyword(s):  
Large Scale ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Systematic Method ◽  
Defective Interfering Particles ◽  
Extracellular Virus ◽  
Transposon Insertion ◽  
Target Sites ◽  
Insertion Sites

Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.

scholarly journals Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication

2001 ◽  
Vol 75 (5) ◽  
pp. 2076-2086 ◽  
Author(s):  
John T. Patton ◽  
Zenobia Taraporewala ◽  
Dayue Chen ◽  
Vladimir Chizhikov ◽  
Melinda Jones ◽  
...  
Keyword(s):  
Cell Culture ◽  
Consensus Sequence ◽  
Active Role ◽  
Serial Passage ◽  
Selective Advantage ◽  
Wild Type ◽  
Reading Frame ◽  
Group A Rotaviruses ◽  
Group A ◽  
Dsrna Genome

ABSTRACT The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.

scholarly journals Replication of Naturally Occurring Woodchuck Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and after Transmission to Naive Woodchucks

2001 ◽  
Vol 75 (8) ◽  
pp. 3811-3818 ◽  
Author(s):  
Mengji Lu ◽  
Gero Hilken ◽  
Dongliang Yang ◽  
Thekla Kemper ◽  
Michael Roggendorf
Keyword(s):  
Hepatitis Virus ◽  
Viral Population ◽  
Core Antigen ◽  
Woodchuck Hepatitis Virus ◽  
Deletion Mutants ◽  
Wild Type ◽  
Lymphoproliferative Response ◽  
Hepatocyte Cultures ◽  
Liver Tissues

ABSTRACT Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.

scholarly journals Luxury at a Cost? Recombinant Mouse Hepatitis Viruses Expressing the Accessory Hemagglutinin Esterase Protein Display Reduced Fitness In Vitro

2005 ◽  
Vol 79 (24) ◽  
pp. 15054-15063 ◽  
Author(s):  
A. Lissenberg ◽  
M. M. Vrolijk ◽  
A. L. W. van Vliet ◽  
M. A. Langereis ◽  
J. D. F. de Groot-Mijnes ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis Virus ◽  
Natural Infection ◽  
Selective Advantage ◽  
Rna Recombination ◽  
Wild Type ◽  
Growth Properties ◽  
Group 2

ABSTRACT Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of “luxury” proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.

scholarly journals Infection of wild-type Autographa californica multicapsid nucleopolyhedrovirus induces in vivo apoptosis of Spodoptera litura larvae

2002 ◽  
Vol 83 (12) ◽  
pp. 3003-3011 ◽  
Author(s):  
Ping Zhang ◽  
Kai Yang ◽  
Xiaojiang Dai ◽  
Yi Pang ◽  
Deming Su
Keyword(s):  
Spodoptera Litura ◽  
Fat Body ◽  
Morphological Changes ◽  
Natural Infection ◽  
Light And Electron Microscopy ◽  
Autographa Californica ◽  
Limiting Factor ◽  
Wild Type ◽  
Budded Virus

Direct evidence of in vivo apoptosis of Spodoptera litura larvae was demonstrated by haemocoel inoculation with wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virus (BV). In sharp contrast to natural infection, cadavers did not melt, liquefy and melanize. Typical morphological changes of apoptosis in insect haemocytes post-infection, including blebbing of the cell surface, chromatin margination and condensation, vacuolization of the cytoplasm and formation of apoptotic bodies, were observed by light and electron microscopy. Total DNAs extracted from virus-infected haemocytes showed DNA ladders. Cleavage of chromatin DNA by endogenous endonucleases were detected in the cells of most tissues cells, including epithelial cells and fat body cells, using terminal dUTP nick end labelling assays. Virogenic stroma and viral nucleocapsids could be seen in the nuclei of a few haemocytes. Yields of BV and OV (occluded virus) produced from the infected S. litura larvae were much lower than from the infected S. exigua larvae. These data suggest that host apoptotic responses to virus infection reduce AcMNPV spread at the level of the organism and that apoptosis could be a host-range limiting factor for baculovirus infections.

scholarly journals STP and Tip Are Essential for Herpesvirus Saimiri Oncogenicity

1998 ◽  
Vol 72 (2) ◽  
pp. 1308-1313 ◽  
Author(s):  
S. Monroe Duboise ◽  
Jie Guo ◽  
Sue Czajak ◽  
Ronald C. Desrosiers ◽  
Jae U. Jung
Keyword(s):  
Cell Culture ◽  
Experimental Infection ◽  
Interleukin 2 ◽  
Common Marmoset ◽  
Herpesvirus Saimiri ◽  
Deletion Mutants ◽  
Wild Type ◽  
Common Marmosets ◽  
Mutant Forms

ABSTRACT Mutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS ΔSTP-C488 and HVS ΔTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.

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