scholarly journals Development of a reverse-genetics system for murine norovirus 3: long-term persistence occurs in the caecum and colon

2012 ◽  
Vol 93 (7) ◽  
pp. 1432-1441 ◽  
Author(s):  
Armando Arias ◽  
Dalan Bailey ◽  
Yasmin Chaudhry ◽  
Ian Goodfellow
Keyword(s):  
Cell Culture ◽  
Capsid Protein ◽  
Reverse Genetics ◽  
Viral Rna ◽  
Viral Gastroenteritis ◽  
Murine Norovirus ◽  
Wild Type ◽  
Adaptive Mutations ◽  
Viral Sequences

Human noroviruses (HuNoV) are a major cause of viral gastroenteritis worldwide, yet, due to the inability to propagate HuNoV in cell culture, murine norovirus (MNV) is typically used as a surrogate to study norovirus biology. MNV-3 represents an attractive strain to study norovirus infections in vivo because it establishes persistence in wild-type mice, yet causes symptoms resembling gastroenteritis in immune-compromised STAT1−/− mice. The lack of reverse-genetics approaches to recover genetically defined MNV-3 has limited further studies on the identification of viral sequences that contribute to persistence. Here we report the establishment of a combined DNA-based reverse-genetics and mouse-model system to study persistent MNV-3 infections in wild-type (C57BL/6) mice. Viral RNA and infectious virus were detected in faeces for at least 56 days after inoculation. Strikingly, the highest concentrations of viral RNA during persistence were detected in the caecum and colon, suggesting that viral persistence is maintained in these tissues. Possible adaptive changes arising during persistence in vivo appeared to accumulate in the minor capsid protein (VP2) and the viral polymerase (NS7), in contrast with adaptive mutations selected during cell-culture passages in RAW264.7 cells that appeared in the major capsid protein (VP1) and non-structural protein NS4. This system provides an attractive model that can be readily used to identify viral sequences that contribute to persistence in an immunocompetent host and to more acute infection in an immunocompromised host, providing new insights into the biology of norovirus infections.

scholarly journals Amino Acid Substitutions in Positions 385 and 393 of the Hydrophobic Region of VP4 May Be Associated with Rotavirus Attenuation and Cell Culture Adaptation

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 408
Author(s):  
Yusheng Guo ◽  
David E. Wentworth ◽  
Karla M. Stucker ◽  
Rebecca A. Halpin ◽  
Ham Ching Lam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Amino Acid ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Nonsynonymous Substitution ◽  
Amino Acid Substitutions ◽  
Viral Gastroenteritis ◽  
Hydrophobic Region ◽  
Hydrophobic Domain ◽  
Underlying Mechanisms

Rotaviruses (RVs) are the leading cause of the acute viral gastroenteritis in young children and livestock animals worldwide. Although live attenuated vaccines have been applied to control RV infection for many years, the underlying mechanisms of RV attenuation following cell culture adaption are unknown. To study these mechanisms at the genomic level, we have sequenced and conducted a comparative analysis of two virulent human (Wa, G1P[8] and M, G3P[8]) and two virulent porcine (Gottfried, G4P[6] and OSU, G5P[7]) RV strains maintained in gnotobiotic piglets for 22, 11, 12 and 9 serial passages, respectively, with their attenuated counterparts serially passaged in MA-104 cell cultures for 25, 43, 54 and 43 passages, respectively. We showed that most of the mutations were clustered in the VP4 gene, with a relatively high nonsynonymous substitution rate (81.2%). Moreover, two amino acid substitutions observed in the VP4 gene were conserved between two or more strain pairs. D385N substitution was found in M, Wa and Gottfried strains, and another one, S471H/L was present in Wa and Gottfried strains. Importantly, D385 was reported previously in another study and may be involved in regulation of virus entry. Of interest, although no 385 substitution was found in OSU strains, the attenuated OSU strain contained a unique D393H substitution within the same VP4 hydrophobic domain. Collectively, our data suggest that the VP4 hydrophobic region may play an important role in RV attenuation and aa385 and aa393 may represent potential targets for RV vaccine development using reverse genetics and site-specific mutagenesis.

scholarly journals Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

2014 ◽  
Vol 89 (1) ◽  
pp. 811-823 ◽  
Author(s):  
Yi-Ping Li ◽  
Santseharay Ramirez ◽  
Lotte Mikkelsen ◽  
Jens Bukh
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adaptive Mutations ◽  
Culture Systems ◽  
Infectious Clones ◽  
Cell Culture Systems ◽  
A Genome

ABSTRACTThe first discovered and sequenced hepatitis C virus (HCV) genome and the firstin vivoinfectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77Cin vivoinfectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5and 104.4FFU/ml, respectively.IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but notin vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developedin vitroinfectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, thein vitroinfectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

scholarly journals Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

2007 ◽  
Vol 81 (23) ◽  
pp. 13168-13179 ◽  
Author(s):  
Artur Kaul ◽  
Ilka Woerz ◽  
Philip Meuleman ◽  
Geert Leroux-Roels ◽  
Ralf Bartenschlager
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Infectious Hepatitis ◽  
Adaptive Mutation ◽  
Viral Genomes ◽  
Adaptive Mutations ◽  
Spread Of Infection

ABSTRACT Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.

scholarly journals Influence of genome-scale RNA structure disruption on the replication of murine norovirus—similar replication kinetics in cell culture but attenuation of viral fitness in vivo

2013 ◽  
Vol 41 (12) ◽  
pp. 6316-6331 ◽  
Author(s):  
Nora McFadden ◽  
Armando Arias ◽  
Inga Dry ◽  
Dalan Bailey ◽  
Jeroen Witteveldt ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Structure ◽  
Viral Fitness ◽  
Murine Norovirus ◽  
Genome Scale

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 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 Characterization of the Glycoprotein Stable Signal Peptide in Mediating Pichinde Virus Replication and Virulence

2016 ◽  
Vol 90 (22) ◽  
pp. 10390-10397 ◽  
Author(s):  
Junjie Shao ◽  
Xiaoying Liu ◽  
Hinh Ly ◽  
Yuying Liang
Keyword(s):  
Cell Culture ◽  
Membrane Fusion ◽  
Viral Entry ◽  
Reverse Genetics ◽  
Cell Entry ◽  
Pichinde Virus ◽  
Viral Virulence ◽  
Stable Signal

ABSTRACTArenaviruses can cause lethal hemorrhagic fevers in humans with few preventative and therapeutic measures. The arenaviral glycoprotein stable signal peptide (SSP) is unique among signal peptides in that it is an integral component of the mature glycoprotein complex (GPC) and plays important roles not only in GPC expression and processing but also in the membrane fusion process during viral entry. Using the Pichinde virus (PICV) reverse genetics system, we analyzed the effects of alanine substitutions at many conserved residues within the SSP on viral replication in cell culture and in a guinea pig infection model. Our data showed that the K33A, F49A, and C57A mutations abolished GPC-mediated cell entry and therefore could not allow for the generation of viable recombinant viruses, demonstrating that these residues are essential for the PICV life cycle. The G2A mutation caused a marked reduction of cell entry at the membrane fusion step, and while this mutant virus was viable, it was significantly attenuatedin vitroandin vivo. The N20A mutation also reduced membrane fusion activity and viral virulence in guinea pigs, but it did not significantly affect cell entry or viral growth in cell culture. Two other mutations (N37A and R55A) did not affect membrane fusion or viral growthin vitrobut significantly reduced viral virulencein vivo. Taken together, our data suggest that the GPC SSP plays an essential role in mediating viral entry and also contributes to viral virulencein vivo.IMPORTANCESeveral arenaviruses, such as Lassa fever virus, can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, and no FDA-approved vaccines or therapies are currently available. Viral entry into cells is mediated by arenavirus GPC that consists of an SSP, the receptor-binding GP1, and transmembrane GP2 protein subunits. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we have shown for the first time in the context of virus infections of cell culture and of guinea pigs that the SSP plays an essential role in mediating the membrane fusion step as well as in other yet-to-be-determined processes during viral infection. Our study provides important insights into the biological roles of GPC SSP and implicates it as a good target for the development of antivirals against deadly human arenavirus pathogens.

scholarly journals Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1989
Author(s):  
Sabine Nitschel ◽  
Luca M. Zaeck ◽  
Madlin Potratz ◽  
Tobias Nolden ◽  
Verena te Kamp ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Rabies Virus ◽  
Point Mutations ◽  
Inverse Correlation ◽  
Virus Release ◽  
Independent Manner ◽  
Adaptive Mutations ◽  
Field Virus

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

scholarly journals Hypervariable Domain of nsP3 of Eastern Equine Encephalitis Virus Is a Critical Determinant of Viral Virulence

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Chetan D. Meshram ◽  
Nikita Shiliaev ◽  
Elena I. Frolova ◽  
Ilya Frolov
Keyword(s):  
Viral Replication ◽  
Capsid Protein ◽  
Host Factors ◽  
Eastern Equine Encephalitis Virus ◽  
Wild Type ◽  
Protein Families ◽  
Host Proteins ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus

ABSTRACT Eastern equine encephalitis virus (EEEV) is the most pathogenic member of the Alphavirus genus in the Togaviridae family. This virus continues to circulate in the New World and has a potential for deliberate use as a bioweapon. Despite the public health threat, to date no attenuated EEEV variants have been applied as live EEEV vaccines. Our previous studies demonstrated the critical function of the hypervariable domain (HVD) in EEEV nsP3 for the assembly of viral replication complexes (vRCs). EEEV HVD contains short linear motifs that recruit host proteins required for vRC formation and function. In this study, we developed a set of EEEV mutants that contained combinations of deletions in nsP3 HVD and clustered mutations in capsid protein, and tested the effects of these modifications on EEEV infection in vivo. These mutations had cumulative negative effects on viral ability to induce meningoencephalitis. The deletions of two critical motifs, which interact with the members of cellular FXR and G3BP protein families, made EEEV cease to be neurovirulent. The additional clustered mutations in capsid protein, which affect its ability to induce transcriptional shutoff, diminished EEEV’s ability to develop viremia. Most notably, despite the inability to induce detectable disease, the designed EEEV mutants remained highly immunogenic and, after a single dose, protected mice against subsequent infection with wild-type (wt) EEEV. Thus, alterations of interactions of EEEV HVD and likely HVDs of other alphaviruses with host factors represent an important direction for development of highly attenuated viruses that can be applied as live vaccines. IMPORTANCE Hypervariable domains (HVDs) of alphavirus nsP3 proteins recruit host proteins into viral replication complexes. The sets of HVD-binding host factors are specific for each alphavirus, and we have previously identified those specific for EEEV. The results of this study demonstrate that the deletions of the binding sites of the G3BP and FXR protein families in the nsP3 HVD of EEEV make the virus avirulent for mice. Mutations in the nuclear localization signal in EEEV capsid protein have an additional negative effect on viral replication in vivo. Despite the inability to cause a detectable disease, the double HVD and triple HVD/capsid mutants induce high levels of neutralizing antibodies. Single immunization protects mice against infection with the highly pathogenic North American strain of EEEV. High safety, the inability to revert to wild-type phenotype, and high immunogenicity make the designed mutants attractive vaccine candidates for EEEV infection.

scholarly journals Protruding Domain of Capsid Protein Is Necessary and Sufficient To Determine Murine Norovirus Replication and Pathogenesis In Vivo

2012 ◽  
Vol 86 (6) ◽  
pp. 2950-2958 ◽  
Author(s):  
D. W. Strong ◽  
L. B. Thackray ◽  
T. J. Smith ◽  
H. W. Virgin
Keyword(s):  
Capsid Protein ◽  
Murine Norovirus ◽  
Necessary And Sufficient
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