Short '1.2× genome' infectious clone initiates deltavirus replication in Boa constrictor cells

Human hepatitis D virus (HDV), discovered in 1977, represented the sole known deltavirus for decades. The dependence on hepatitis B virus (HBV) co-infection and its glycoproteins for infectious particle formation led to the assumption that deltaviruses are human-only pathogens. However, since 2018, several reports have described identification of HDV-like agents from various hosts but without co-infecting hepadnaviruses. Indeed, we demonstrated that Swiss snake colony virus 1 (SwSCV-1) uses arenaviruses as the helper for infectious particle formation, thus shaking the dogmatic alliance with hepadnaviruses for completing deltavirus life cycle. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used SwSCV-1 as the model to compare the ability of the previously reported "2× genome" and the "1.2× genome" plasmid constructs/infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. We hypothesize that duplicating the ribozymes facilitates the cleavage of genome multimers into unit-length pieces during the initial round of replication. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.

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Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

Viruses ◽

10.3390/v14010107 ◽

2022 ◽

Vol 14 (1) ◽

pp. 107

Author(s):

Leonora Szirovicza ◽

Udo Hetzel ◽

Anja Kipar ◽

Jussi Hepojoki

Keyword(s):

Cell Culture ◽

Infectious Clone ◽

Helper Virus ◽

Northern Blotting ◽

Cellular Interactions ◽

Hepatitis D ◽

Boa Constrictor ◽

Infectious Clones ◽

In Vitro Systems

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported “2× genome” and the “1.2× genome” infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.

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

Journal of Virology ◽

10.1128/jvi.02877-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 811-823 ◽

Cited By ~ 32

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.

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Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation

Journal of General Virology ◽

10.1099/vir.0.81958-0 ◽

2006 ◽

Vol 87 (8) ◽

pp. 2263-2268 ◽

Cited By ~ 26

Author(s):

Dennis J. Pierro ◽

Ma Isabel Salazar ◽

Barry J. Beaty ◽

Ken E. Olson

Keyword(s):

Cell Culture ◽

Dengue Virus ◽

Virus Type ◽

Mammalian Cells ◽

Infectious Clone ◽

Cell Types ◽

Dengue Virus Type 2

A full-length infectious cDNA clone (ic) was constructed from the genome of the dengue virus type 2 (DENV-2) Jamaica83 1409 strain, pBAC1409ic, by using a bacterial artifical chromosome plasmid system. Infectious virus was generated and characterized for growth in cell culture and for infection in Aedes aegypti mosquitoes. During construction, an isoleucine to methionine (Ile→Met) change was found at position 6 in the envelope glycoprotein sequence between low- and high-passage DENV-2 1409 strains. In vitro-transcribed genomic RNA of 1409ic with E6-Ile produced infectious virions following electroporation in mosquito cells, but not mammalian cells, while 1409ic RNA with an E6-Met mutation produced virus in both cell types. Moreover, DENV-2 1409 with the E6-Ile residue produced syncytia in C6/36 cell culture, whereas viruses with E6-Met did not. However, in vitro cell culture-derived growth-curve data and in vivo mosquito-infection rates revealed that none of the analysed DENV-2 strains differed from each other.

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Microfrabricated in-vitro cell culture systems for investigating cellular interactions: fabricating a model system for cardiac myocytes

10.1117/12.379567 ◽

2000 ◽

Author(s):

Jennifer Deutsch ◽

Tejal A. Desai ◽

Delara Motlagh ◽

Brenda Russell

Keyword(s):

Cell Culture ◽

Cardiac Myocytes ◽

Model System ◽

Cellular Interactions ◽

Culture Systems ◽

In Vitro Cell Culture ◽

Cell Culture Systems

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Ebola Virus VP40 Late Domains Are Not Essential for Viral Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.79.16.10300-10307.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10300-10307 ◽

Cited By ~ 61

Author(s):

Gabriele Neumann ◽

Hideki Ebihara ◽

Ayato Takada ◽

Takeshi Noda ◽

Darwyn Kobasa ◽

...

Keyword(s):

Cell Culture ◽

Virus Replication ◽

Reverse Genetics ◽

Ebola Virus ◽

Critical Role ◽

Particle Formation ◽

Late Domain ◽

The Matrix ◽

Late Domains

ABSTRACT Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.

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Inhibition of p38 MAPK by SCIO-469 Suppresses TNF Generation and Promotes CD34+ Cell Survival in an In Vitro MDS Cell Culture Model.

Blood ◽

10.1182/blood.v104.11.3424.3424 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3424-3424

Author(s):

Tony A. Navas ◽

Aaron N. Nguyen ◽

Jing Y. Ma ◽

Elizabeth G. Stebbins ◽

Edwin Haghnazari ◽

...

Keyword(s):

Cell Culture ◽

Bone Marrow ◽

Cell Line ◽

P38 Mapk ◽

Specific Inhibitor ◽

Cellular Interactions ◽

Cell Culture Model ◽

Culture Model ◽

Inhibitory Molecules

Abstract Progress in the development of more effective therapeutics for myelodysplastic syndrome (MDS) has been limited by the lack of targets critical to the pathobiology of the disease. Ineffective hematopoiesis in MDS is characterized by accelerated proliferation and premature apoptotic death of progenitors and their progeny that is potentiated by the local generation of inhibitory molecules, including TNFa, TGFß, FasL, and VEGF. To identify upstream regulatory signals that may coordinate activation of inhibitory molecules, we used an in vitro cell culture model incorporating a CD34+ MDS cell line isolated from a RAEB-t patient, normal bone marrow stromal cells (BMSC), and/ or bone marrow mononuclear cells (BMMNC) to determine effects of cell-cell interactions on secretion of inhibitory hematopoietic cytokines. The role of p38 MAP kinase, a regulatory kinase involved in the convergence of inhibitory cytokine activation and signaling, was evaluated in this interaction. We found that p38 MAPK is induced under basal culture conditions in the MDS cell line and is further activated by TNFa or TGFß. In all cases, p38 activation was reduced by SCIO-469, a potent and specific inhibitor of p38a activity. SCIO-469 does not directly block p38 activation, suggesting a feedback loop is interrupted when p38 kinase activity is inhibited in MDS cells. To determine the effects of cellular interactions, the MDS cell line was co-cultured with either BMSC, BMMNCs or both from normal donors, and TNFa and FasL secretion were measured after 3 days incubation. TNFa and FasL were detected in culture supernatants when the MDS cell line was co-cultured with BMMNC but not when co-cultured with BMSC. TNFa secretion by BMMNCs was dependent on MDS cell contact and was significantly inhibited by SCIO-469. The addition of BMSC to the MDS and BMMNC co-culture prevented TNFa elevation, suggesting BMSCs as a dominant source for anti-inflammatory signal(s). VEGF, FGF-ß, TGFß2, BDNF, TIMP-1, TIMP-2 and IL-6 secretion by BMSC was induced by MDS co-culture, whereas SCIO-469 blocked cytokine induction. To determine the effects of SCIO-469 and MDS clone-induced BM cytokine secretion on normal CD34+ proliferation, we co-cultured BMMNCs and BMSC in transwell inserts in the presence or absence of the MDS cell line with or without SCIO-469. CD34+ proliferation was assessed in cells cultured in outer wells. CD34+ progenitors proliferated in culture at the same rate as those co-cultured with BMSC, BMMNC and MDS for 6 days. At longer intervals, viability of progenitors cultured with the MDS line declined, whereas treatment with SCIO-469 abrogated the decrease in CD34+ viability. These results implicate p38a as a critical target in the induction of pro-apoptotic cytokines in MDS, and that selective inhibition of p38 by SCIO-469 may provide a novel therapeutic strategy for MDS.

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The 164 K, 165 K, and 167 K residues of VP1 are vital for goose parvovirus proliferation in GEFs based on PCR-based reverse genetics system

Virology Journal ◽

10.1186/s12985-019-1237-2 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 1

Author(s):

Peng Liu ◽

Liqin Yang ◽

Jingyue Zhang ◽

Tao Wang ◽

Yuanyuan Wu ◽

...

Keyword(s):

Nuclear Import ◽

Reverse Genetics ◽

Clinical Sample ◽

Infectious Clone ◽

Progeny Virus ◽

Infectious Clones ◽

Goose Parvovirus ◽

Growth Character ◽

Full Length Genome

Abstract Background Goose parvovirus (GPV) is the etiological agent of Derzsy’s disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro. Methods In this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17–22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged. Results The rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus. Conclusions The rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.

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Cell Culture Models for the Study of Hepatitis D Virus Entry and Infection

Viruses ◽

10.3390/v13081532 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1532

Author(s):

Margaux J. Heuschkel ◽

Thomas F. Baumert ◽

Eloi R. Verrier

Keyword(s):

Cell Culture ◽

Sodium Taurocholate ◽

Chronic Viral Hepatitis ◽

Experimental Models ◽

Model Systems ◽

Hepatitis D ◽

Hepatitis D Virus ◽

Culture Models ◽

Cell Culture Models

Chronic hepatitis D is one of the most severe and aggressive forms of chronic viral hepatitis with a high risk of developing hepatocellular carcinoma (HCC). It results from the co-infection of the liver with the hepatitis B virus (HBV) and its satellite, the hepatitis D virus (HDV). Although current therapies can control HBV infection, no treatment that efficiently eliminates HDV is available and novel therapeutic strategies are needed. Although the HDV cycle is well described, the lack of simple experimental models has restricted the study of host–virus interactions, even if they represent relevant therapeutic targets. In the last few years, the discovery of the sodium taurocholate co-transporting polypeptide (NTCP) as a key cellular entry factor for HBV and HDV has allowed the development of new cell culture models susceptible to HBV and HDV infection. In this review, we summarize the main in vitro model systems used for the study of HDV entry and infection, discuss their benefits and limitations and highlight perspectives for future developments.

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Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010005161x ◽

1974 ◽

Vol 32 ◽

pp. 320-321

Author(s):

J. P. Revel

Keyword(s):

Developmental Stages ◽

Three Dimensional ◽

Cell Movement ◽

Cellular Interactions ◽

Serial Sections ◽

Cell Sheets ◽

Freeze Etching ◽

Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

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